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MSU Is An AIIirrndIve ActIorVEquel Opportunity Inetltulon
emails-o:

INVESTIGATION OF DYSMYELINOGENESIS
IN CAPRINE B-MANNOSIDOSIS:
BIOCHEMICAL, CELL CULTURE,
MORPHOLOGICAL, AND ENDOCRINE STUDIES

By

Philip Joseph Boyer

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Pathology

1989 .

ABSTRACT
INVESTIGATION OF DYSMYELINOGENESIS
IN CAPRINE B-MANNOSIDOSIS:
BIOCHEMICAL, CELL CULTURE,
MORPHOLOGICAL, AND ENDOCRINE STUDIES
By

Philip Joseph Boyer

Central nervous system (CNS) myelin deficiency is a consistent
pathological feature of caprine B-mannosidosis, an autosomal recessive
neurovisceral lysosomal storage disease. The four projects presented in this
dissertation were designed to examine some of the factors which could contribute
to the myelin deficiency found in affected animals.

Quantification of regional central nervous system oligosaccharide
accumulation addressed the possibility of an association between the storage of
oligosaccharides and myelin deficits. Results indicate that the extent of regional
CNS accumulation of oligosaccharide is not associated with regional differences
in severity of myelin deficiency in caprine B-mannosidosis.

L Cell culture studies examined oligodendrocytes from affected and control
animals to compare their number, morphology, and immunostaining
characteristics and assess the possibility of intrinsic oligodendrocyte defects.
Results indicate that differentiated oligodendrocytes from affected animals do not
show morphological abnormalities in culture. However, increased numbers of
galactocerebroside-negative bipolar cells, which may be glial progenitor cells,

were present in affected animal cultures, suggesting the possibility of a defect in

differentiation to mature oligodendrocytes, with persistence of the
undifferentiated glia during late stages of development.

Astrocyte changes at various stages of myelination of the optic nerve were
examined by in__v_i1Q immunocytochemical studies to assess developmental
features of astrocytic abnormalities. Results suggest that astrocyte changes are
present in the affected animal optic nerve even during early stages of myelination.
and that changes are not progressive. The increased density of astrocyte processes
appears to be due to a greater number of processes extending from astrocytes,
although the possibility of an increased number of astrocytes with redistribution
of glial fibrillary acidic protein from cell bodies into processes has not been ruled
out.

Examination of thyroid morphology and function revealed that extensive
and developmentally progressive morphological abnormalities as well as
statistically significant thyroid function deficits are present in affected goats. Thus,
a role for reduced thyroid hormone levels in hypomyelination in caprine B-

mannosidosis is possible.

ACKNOWLEDGEMENTS

I sincerely thank both Dr. Kathryn Lovell and Dr. Margaret Jones for.
their guidance, support, and encouragement throughout my three years of
research. I also wish to thank Dr. Adalbert Koestner and Dr. James Trosko for
their work as members of my Ph.D. committee, Ralph Common for the
photographic printing in the thesis, and Robert Kranich for help with various
aspects of this work. Additional acknowledgements follow various sections of this

dissertation.

iv

TABLE OF CONTENTS

List of Tables 1 vi
List of Figures vii
Introduction 1

Regional central nervous system oligosaccharide

storage in caprine a-mannosldosis 28

Investigation of dysmyeilnogenesis in caprine
s-mannosidosis: in vitrg characterization

of Oligodendrocytes . 46

Astrocyte abnormalities in caprine s-mannosidosls:

an immunocytochemical analysis of optic nerve 68

Caprine s-mannosidosis: abnormal thyroid structure

and function in a lysosomal storage disease 90

LIST OF TABLES

Caprine s-mannosidosis: abnormal thyroid structure and function in a lysosomal storage -
disease

Table 1 117 Mean thyroid weights at various stages of development

Table 2 118 Thyroid hormone assay summaries from six affected and control animal

age-matched pairs

vi

LIST OF FIGURES

Ely-.11: M _99_e__eFl r Tit!
Introduction
Figure 1 27 Summary of known and possible connections between the genetic defect in s-

mannosidosls and decreased myelinatlon.

Regional central nervous system oligosaccharide storage In caprine n-mannosidosis

Figure 1 43 Thin layer chromatography of oligosaccharide standards, CH white matter crude
tissue extracts, and Bio-Gel P—2 isolated oligosaccharides.

Figure 2 45 Regional CNS DS and TS content In affected animals.

Figure 3 45 Regional CNS s—mannosidase activity in control animals.

Investigation of dysmyelinogenesis In caprine a-mannosidosis: In vit_rg characterization

of oligodendrocytes

Figure 1 65 Control and affected animal cells after 3 days in culture In Petri plates.

FigUre 2 65 Phase contrast and GC immunofiuorescence-Iabeiled. from the same field, of
control and affected animal cells 3 days after plating of floating cells on poly-
L-lysine coated glass coverslips.

Figure 3 67 Control and affected animal cells between 12 and 21 days on coverslips labelled
by GC immunofluorescence.

Figure 4 67 Control and affected animal cells at 8 days on coverslips.

vii

Fig. i Pg. fl Eigurg Titlg

Astrocyte abnormalities in caprine s-mannosidosis: an immunocytochemical analysis of

optic nerve

Figure 1 86 GFAP-labelled. Epon-embedded optic nerve sections from age-matched control
and affected goats at 115/150 days gestation. 3 days postnatal and 4 weeks
postnatal.

Figure 2 87 GFAP reaction product density determined by image analysis In age-matched
control and affected animal optic nerves.

Figure 3 89 GFAP-labelled 60 um Vibratome sections of optic nerve from 3-day-old control

and affected goats.

Caprine a-mannosidosis: abnormal thyroid structure and function In a lysosomal storage

disease

Figure 1 120 Light micrographs of toluidine blue stained 1um Epon sections from control and
affected animals at 96/150 days gestation. 124/ 150 days gestation, and 3 days
of age.

Figure 2 122 Electron micrographs of thyroid follicular cells from 3—day-old control (A) and
affected goats.

Figure 3 124 T3 and T, release pattern assessed in serum samples drawn at 15 minute
intervals over 4-hours, between 8:00 am and 12:00 noon, from a representative
herd A (normal animals) and herd 8 (carriers of s-mannosidosis) animal

Figure 4 124 T, (A) and Ta (8) mean values from blood draws at birth (day 0) and between
days 1 and 14 postnatal from herds A (n = 5) and B (n = 10. days 0-8; n = 5,
days 9-14).

viii

INTRODUCTION

2

In caprine Bomannosidosis, an autosomal recessive disease of glycoprotein
metabolism (28), deficient activity of the lysosomal enzyme B-mannosidase (EC
3.2.1.25) is associated with tissue and body fluid accumulation of the enzyme’s
putative substrates, including a large amount of a trisaccharide (TS, mannosle 1-
4N-acetylg1ucosaminy181-4N-acetylglucosamine), a smaller amount of a.
disaccharide (DS, mannosle1-4N-acetylglucosamine), and much smaller
quantities of more complex oligosaccharides (19,29,30,38,39,47,48). Clinical and
pathological abnormalities in affected goats include facial dysmorphism;
neurological signs include deafness, ataxia, and tremor; neurovisceral cytoplasmic
vacuolation; and central nervous system hypomyelination (37,41).

B-Mannosidase is one of many enzymes involved with catabolism of the
oligosaccharide portion of glycoproteins. One or both of two enzymes can cleave
N-linked oligosaccharides from glycoproteins, a step which precedes
oligosaccharide degradation. An endo-B-N-acetylglucosaminidase can cleave the
chitobiosyl GlcNAcBlAGlcNAc bond while an endo-B-aspartylglucosaminidase
can cleave the GlcNAcBl-lasparagine bond. The predominant storage of TS and
secondary storage of DS in goats affected with B~mannosidosis suggests that
endo-B-aspartylglucosaminidase activity predominates but that significant endo-
B-N-acetylglucosaminidase activity is present too (30). Once the glycoprotein is
cleaved, the oligosaccharide unit is sequentially degraded in a series of enzyme-
mediated steps (21). B-Marmosidase activity act on the last step of the catabolic
scheme and cleaves a 81-4 linkage between mannose and N -acetyl-glucosamine.

Two forms of B-mannosidase exist (56), a cytoplasmic form that appears to be

3

localized exclusively in caprine liver, and a lysosomal form that has been found
in all tissues assayed for it. The cytoplasmic form of the enzyme is deficient in
animals affected with B-mannosidosis (58), and will be referred to here as B-
mannosidase. Deficient B-mannosidase activity leaves the enzyme’s substrates
uncatabolized, and lysosomal storage of the substrates is thought to explain the.
cytoplasmic vacuolation seen in most cell types of affected animals, although non-
lysosomal cytoplasmic storage and extracellular storage may also be present. The
presence of oligosaccharides in urine from affected animals and in affected
animal fibroblast cell culture media indicates that oligosaccharides exist outside
of lysosomes as well as inside them. In fact, the TS:DS ratio is nearly equal in
urine and cell culture media, compared to the TS predominance in tissue,
indicating a greater diffusion of DS out of lysosomes, probably consistent with the
smaller molecular size of DS.

A caudal-to-rostral pattern of increasingly severe central nervous system
(CNS) myelin deficiency is a major pathological feature of caprine 8-
mannosidosis. In affected goats, the spinal cord, which is myelinated relatively
early in development, has a moderate degree of myelin deficiency while the --
cerebral hemispheres (CH), which are myelinated later in development, have a
severe deficiency of myelin. Both quantitative (43) and qualitative observations
(44,45) have suggested that oligodendrocyte number is reduced in regions where
myelin deficiency is present. Other in Lit/.0 observations include: (a) a greater
proportion of oligodendrocytes with vacuoles and dark cytoplasm in affected

goats, beginning prior to development in corpus callosum (Lovell, in

4

preparation); (b) no ultrastructural difference in CNS myelin sheaths between
affected and control animals; (c) no evidence for defective axon-oligodendrocyte
interaction; and (d) absence of either oligodendrocyte or myelin degeneration
(37,44,45). The possibility that affected animal oligodendrocytes produce
proportionally fewer myelin sheath segments than control animal.
oligodendrocytes has been suggested but not definitively addressed. Taken
together, these findings suggest that, in affected animals, (a) myelination is
disrupted during development and (b) some alteration in oligodendrocyte
proliferation, differentiation, and/or function is present.

Oligodendrocytes are glial cells which produce and maintain CNS myelin.
Recent evidence from mixed glial cell cultures from rat optic nerve suggests that
oligodendrocytes and type II astrocytes arise from a common progenitor cell (55).
As compared to Schwann cells in the peripheral nervous system, where a single
cell contributes myelin to a single axon, CNS white matter oligodendrocytes
contribute myelin internodes to as many as 50 axons. Oligodendrocytes are also
found in the gray matter, where they are called "satellite oligodendrocytes."
Oligodendrocytes develop through a maturation scheme which progresses, as seen
ultrastructurally, from light in appearance with abundant cytoplasm and a large
nucleus to dark in appearance with scanty cytoplasm and a compact nucl'eus. At
all stages of maturation, prominent cytoplasmic Golgi and endoplasmic reticulum
content is present in oligodendrocytes.

No good explanation is yet formulated to explain the pathogenesis of the

unique pattern of hypomyelination in caprine B-mannosidosis. Figure 1

5

summarizes what is known and not known about the connection between the
genetic defect in caprine B-mannosidosis and hypomyelination. Solid arrows
indicate connections that have been established while dashed arrows indicate
uncertain connections. The fundamental genetic defect which leads to deficient
enzyme activity in goats is likely to be restricted to the B-mannosidase locus. If .
so, the pathogenesis of abnormalities present in B-mannosidosis, including
deficient myelination, would be directly or indirectly linked to deficient activity
of B-mannosidase. At the cellular level, possible explanations of direct effects of
deficient B-mannosidase activity on oligodendrocytes include (1) a toxic,
inhibitory, or space-occupying effect of oligosaccharide accumulation, and (2)
effects due to altered glchprotein metabolism, possibly through a toxic
intermediate from an alternate metabolic pathway. Alternatively, effects on
oligodendrocytes may be secondary to effects of deficient B-mannosidase activity
on another organ, for instance the thyroid, which would have implications for
oligodendrocyte development and function. Less likely, but still possible, are
efl'ects of the genetic defect other than on the B-mannosidase locus that could
have a direct or indirect effect on oligodendrocytes.

Clinical manifestations of B-mannosidosis are less severe in humans than
in goats. Of the 5 cases of human B-mannosidosis described in the literature, all
had mental insufficiency and 3 had some degree of hearing and speech
impairment, however, none showed neurological signs consistent with gross
myelin deficiency (18,24,71). Computerized tomography scan of one patient found

no lesions indicative of myelin deficiency (18). The difference in oligosaccharide

6

storage in human and caprine B-mannosidosis provides a possible explanation of
the differences in phenotypic expression. Of potential importance, DS is the only
oligosaccharide detected in the urine of human B-mannosidosis patients
(17,24,71), sharply contrasting with the TS storage predominance in caprine B-
mannosidosis. However, the results of the oligosaccharide study in this.
dissertation suggest that extent of oligosaccharide accumulation is not associated
with the degree of myelin deficiency. Thus, the differences in oligosaccharide
storage between caprine and human B-mannosidosis probably do not help to
explain the species differences in myelination.

With respect to other neurovisceral lysosomal storage diseases, a -
mannosidosis, associated with deficient activity of lysosomal a-mannosidase,
another enzyme in the N-linked oligosaccharide catabolic pathway, is the most
closely related to B-mannosidosis. Many similarities in CNS pathology, especially
with respect to the presence of axonal spheroids and the distribution of
cytoplasmic vacuolation exist between a-mannosidosis and B-mannosidosis (44).
However, central nervous system myelin deficiency is not an early feature of
bovine (35,36) and human a-mannosidosis (50,61), although it has been reported
in feline a-mannosidosis at 33 days of age and beyond (67). Thus, the factor or
factors that cause myelin deficiency in B-mannosidosis are probably distinct from
general CNS storage of oligosaccharides or storage of the specific complex
oligosaccharides in a-mannosidosis.

Oligodendrocyte deficiency is a major feature of several dysmyelinating

disorders including the jimpy mouse (49,60), the myelin-deficient rat (23), and the

7
twitcher mouse (63). The etiology of oligodendrocyte deficiency in each of these
diseases appears to be quite different from that in 8-mannosidosis. For instance,
caprine B-mannosidosis differs in 2in from both twitcher and jimpy mutants in
several ways but, most importantly, no evidence of oligodendrocyte degeneration
has been seen in B-mannosidosis (44,45), although degeneration is a major,
feature .in the twitcher mouse (62) and jimpy mouse (40). Cell culture studies
in the jimpy mouse (10) and the twitcher mouse (51) have found relatively
normal numbers and morphological characteristics of oligodendrocytes in their
early stages of development but abnormalities become apparent within 7 to 10
days of culture. Abnormalities included degeneration of oligodendrocytes in
twitcher mice and decreased numbers and altered morphologic features and
immunofluorescent labeling in jimpy mice, features consistent with what is found
in 2in in the respective disorders. As presented in this dissertation, in 211.132
characteristics of oligodendrocytes isolated from animals affected with 8-
mannosidosis that contrast with the in mm findings in twitcher and jimpy
include: (1) no evidence of cell degeneration, (2) no evidence of morphological
and immunolabelling abnormalities, and (3) the presence of increased numbers
of bipolar cells, possibly glial progenitor cells. While it is important to note that
the caprine oligodendrocyte cultures were initiated with relatively mature cells,
compared to the immature cells studied in the mouse myelination mutant
cultures, the mechanism of dysmyelination is different in caprine B-mannosidosis

than in twitcher or jimpy mice.

8

Astrocytes respond to central nervous system (CNS) pathological changes
with "gliosis.” Gliosis consists of (1) astrocyte proliferation and/or (2) astrocyte
hypertrophy, with an increased number of extended processes and an increased
expression of the astrocyte-specific intermediate filament glial fibrillary acidic
protein (GFAP) (15,42). Gliosis has been described in a variety of CNS
pathological conditions including trauma, inflammatory diseases, hepatic
encephalopathy, degenerative diseases, and aging (15,27,31,68). Additionally,
gliosis has been identified in the CNS of various myelination mutant mice,
including jimpy, quaking, shiverer, and mld mutants (11). Of considerable
interest, findings similar to those seen in the optic nerve in B-mannosidosis have
been identified in various CNS regions in the jimpy mouse. Gliosis in various
CNS regions preceding myelination or in early stages of myelination was recently
reported in the jimpy mouse and was attributed to glial cell hypertrophy and not
to an increased number of astrocytes (12).

Thyroid hormones play an important role during the development of the
central nervous system (1,32,33) and considerable evidence indicates that thyroid
hormones have a direct impact on myelination (33,69,70). For example, fetal
sheep from experimentally hypothyroid ewes have a decreased number of cells,
presumably including oligodendrocytes, and deficient myelination, and both
situations are reversible by injection of ewes with iodized oil (52). In the rat,
thyroxine deficiency leads to myelin deficiency (6, 59). Additionally, cell culture
studies have shown that thyroid hormones are important for the differentiation

and function of oligodendrocytes (2). In general, hyperthyroidism has been

9
associated with early initiation and premature termination of myelination while
hypothyroidism has been associated with deficient myelination (69). In summary,
both in xiyg and in mm studies have shown quite clearly that thyroid hormones
have significant effects on oligodendrocyte differentiation and function
(2,6,13,14,16,20,46,53,59).

Recent evidence in sheep, an animal similar to goats with respect to
central nervous system development, indicates that decreased thyroid hormone
levels may play a causative role in the hypomyelination of border disease (3).
Severe central nervous system hypomyelination, involving spinal cord, brainstem,
and cerebral hemispheres, is a major pathological feature of border disease, a
disease of sheep caused by in mm infection of fetuses with the border disease
virus (BDV) (5.7-9.54). A reduced thyroid hormone level, possibly secondary to
follicular cell invasion by BDV, has been postulated to be the causative factor
in the severe CNS myelin deficiency (3). Considerable similarity exists between
border disease and B-mannosidosis. Average T, and T. levels in border disease
sheep were 60% and 74% of control values, respectively, similar to the extent of
reduction in B-mannosidosis. Clinical features of border disease which have
similarities to features of caprine B-mannosidosis (37,41) include skeletal
abnormalities such as doming of the skull, small and abnormally placed orbits,
and abnormalities of limb bones (4,7,66), and neurological signs including spasm,
tremor, and locomotor defects (8). In fetal sheep from ewes with experimentally
induced iodine deficiency, doming of the skull, retrognathia, subluxation of the

front joints, and hypomyelination are prominent morphological abnormalities and

10
each of these features can be fully or partially reversed after administration of
iodine to the ewe at 100/ 150 days gestation (53). Thus, even though the etiology
of B-mannosidosis, border disease, and prenatal iodine deficiency are distinctly
different, it is possible that a common pathogenic mechanism of hypomyelination
and other phenotypic features is reduced thyroid hormone levels secondary to.
thyroid dysfunction.

Several lines of evidence suggest that B-mannosidase plays an important
role in thyroid metabolism. First, thyroglobulin is the predominant glycoprotein
in the thyroid and its degradation includes lysosomal catabolism of N-linked
oligosaccharides (34,64,72), requiring B-mannosidase activity. Second, control
goat thyroid B-mannosidase activity is the highest of any organ tested
(unpublished data, and (52)). Third, large quantities of oligosaccharides have
been isolated and characterized from thyroids of affected goats (29).

Thyroglobulin is a glycoprotein containing a ManBlAGlcNAc residue in
its N-linked oligosaccharides (64,72). The direct impact of B-mannosidase
deficiency on thyroglobulin metabolism would occur during lysosomal catabolism
of thyroglobulin. It follows that a thyroglobulin degradation impediment or defect
is the most likely alteration that would lead to decreased thyroid hormone
secretion in B-mannosidosis. Absence of affected goat lysosomal B-mannosidase
activity and accumulation of oligosaccharides could have an adverse effect on
thyroglobulin degradation, and then on thyroid hormone release, through (a)
decreased availability of primary lysosomes due to impaired recycling of

secondary lysosomes (22,57) , and (b) interference in cell function by storage

11

vacuole accumulation. Relevant to both possibilities, a recent study identified a
possible post-endocytosis regulation step in thyroglobulin catabolism, where there
is a lag period between fusion of endosomes with primary lysosomes to form
secondary lysosomes (57). Alterations in the availability of primary lysosomes,
due to alterations in synthesis or in accessibility, might adversely affect post-_
endocytosis regulation. Thyroid metabolic alterations other than disruption of
catabolism--such as a thyroglobulin synthesis defect, an organification defect, a
hormone receptor abnormality, or a thyrotropin deficiency--are much less likely
to be involved in the thyroid hormone deficits in B-mannosidosis. It is possible,
however, that metabolic perturbations secondary to abnormal glycoprotein
metabolism could adversely affect thyroglobulin metabolism.

The four separate research projects presented here were designed to
explore some of those factors which could have an impact on abnormal
oligodendrocyte development and hypomyelination in caprine B-mannosidosis. In
the first project, quantitation of regional CNS oligosaccharide accumulation
addressed the possibility of an association between the stored oligosaccharides
and myelin deficits. Results indicate that the extent of regional CNS
accumulation of TS and DS is not associated with regional differences in severity
of myelin deficiency in caprine B-mannosidosis. Thus, it is unlikely that
oligosaccharide accumulation plays a pathogenetic role in hypomyelination.

In the second project, oligodendrocytes from affected and control animals
were examined in £1319 to compare their numbers, morphology, and

immunostaining characteristics and assess the possibility of intrinsic

12
oligodendrocyte defects. Results from the cell culture study are consistent with
in ijo morphological observations and suggest that differentiated
oligodendrocytes from affected animals do not show morphological abnormalities
in culture. However, increased numbers of galactocerebroside-negative bipolar
cells, which may be glial progenitor cells, were present in affected animal.
cultures. Morphologically similar cells have been identified as glial progenitor
cells in mixed glial cultures from neonatal rats (55,65) and fetal sheep (26), using
anti-galactocerebroside and anti-A,B,, a monoclonal antibody directed against a
complex ganglioside (25). Presence of the bipolar cells in increased numbers in
affected animals, if they indeed are glial progenitor cells, suggests the possibility
of a defect in differentiation to mature oligodendrocytes, with persistence of the
undifferentiated glia during late stages of development. Additional studies are
needed to further investigate bipolar cells and to determine the role of
impairment of oligodendrocyte development in hypomyelination in caprine B-
mannosidosis. Studies need to include, initially, full documentation of the bipolar
cells with anti-A,B, and possibly with fluorescent microbeads. A,B,-positive
labeling would identify the cells as glial progenitor cells while microbead uptake
would identify the cells as macrophages. Additionally, assuming that the cells are
glial progenitor cells, isolation of bipolar cells from affected animal CNS and
experimentation with addition of various factors -- e.g. thyroid hormone and
nerve growth factors -- to see if an actual block in differentiation is present

would be of interest.

13

In the third project, astrocyte changes at various stages of myelination of
the optic nerve were addressed by 194/110 immunocytochemical studies. This
study suggests that astrocyte changes are present in the optic nerves of affected
animals even during early stages of myelination and that changes are not
progressive. Increased astrocyte process density at a time early in myelination
suggests that gliosis may not be simply a reaction to a decreased number of
myelin sheaths but may take place in response to other abnormalities, possibly
in response to a decreased number or abnormal development of oligodendrocytes
or perturbations in oligosaccharide metabolism. Additionally, the increased
density of GFAP+ astrocyte processes appears to be due to a greater number of
processes extending from astrocytes, althOugh the possibility of an increased
number of astrocytes with redistribution of GFAP from cell bodies into processes
has not been ruled out. Further examination of the gliosis, in spinal cord,
cerebellum, and corpus callosum, will help to define the extent of the change and
allow assessment of the association between gliosis and regional variations in
myelin deficiency. Additionally, examination of the corpus callosum will allow for
examination of white matter before myelination has begun and then will allow
for examination of the issue of whether gliosis is present before myelination
begins.

In the fourth project, examination of morphology and function of the
affected animal thyroid was designed to assess whether decreased hormone
secretion could be a factor leading to impaired myelination. The thyroid study

documents the presence of extensive and developmentally progressive

14

morphological abnormalities in the thyroid of goats affected with B-mannosidosis.
Additionally, evidence suggesting deficient thyroid function in affected goats is
presented. This functional abnormality may be due, at least in part, to the
extensive thyroid follicular cell morphological abnormalities or to metabolic
perturbations secondary to deficient B-mannosidase activity. A role for reduced
thyroid hormone levels in the hypomyelination and other clinical and pathological
abnormalities seen in goats affected with B-mannosidosis is possible and needs
further investigation.

Since thyroid hormone deficiency appears to be a strong candidate for
explaining hypomyelination in B-mannosidosis, further examination of the
relationship between thyroid hormone deficiency and hypomyelination are in
order. The following hypothesis may help explain the role deficient thyroid
hormone levels may play in the disruption of myelination in B—mannosidosis. At
the onset of thyroid function, at between 50 and 60 days gestation, thyroid
hormone levels may be normal, or at least sufficient to support relatively normal
myelination. However, as development proceeds, with continual degradation of
thyroglobulin and progressive accumulation of uncatabolized oligosaccharides
within lysosomes, progressively greater disruption of thyroid function may result.
Progressively decreased amounts of thyroid hormone over time could lead to
increasingly! severe myelin deficiency in later myelinating regions. Both (a)
substantial morphological alterations present as early as 96/ 150 days gestation
and (b) documentation of the progressive nature of accumulation during

development offer circumstantial support for the hypothesis. Evaluation of

15

thyroid hormone levels at various stages of in glam development of affected and
control fetal animals will help assess the possibility of a role for thyroid hormone
deficiency in the hypomyelination of B-mannosidosis. Clearly, further investigation
of thyroid function in B-mannosidosis in warranted. Further work could include
(a) verification of the current thyroid function findings in both fetal and neonatal,
affected animals, (b) assessment of affected and control animal thyroid colloid
immunocytochemically, (c) assessment of anterior pituitary morphology at both
the light and electron microsc0pic levels, and (d) characterization of degree of
vacuolation and secretory granule content in anterior pituitary thyrotrophs
ultrastructurally and possibly immunocytochemically. The absence of a thyroid
stimulating hormone (TSH) assay that works in goats limits our ability to
measure this important parameter. However, it may be worthwhile to attempt to
stimulate thyroid hormone secretion by injection of affected and control goats
with TSH releasing hormone (TRH) to challenge their thyroid secretory capacity.

Four major projects were carried out as part of this dissertation in an
attempt to help define the pathogenesis of myelin deficiency in B-mannosidosis
(Fig. 1). The two most promising results were (a) the finding of an increased
number of bipolar cells, possibly glial progenitor cells, in affected animal white
matter cultures and (b) the documentation of deficient thyroid function in
affected animals. The increased number of bipolar cells may indicate that a block
in differentiation of progenitor cells to mature oligodendrocytes is present in
affected animals. Deficient thyroid hormone levels in affected animals may help

explain both blocked differentiation and abnormal function. This is the first

16
report suggesting that the pathogenesis of CNS lesions in a storage disease could
involve endocrine perturbations. Thus, the current findings may underscore the
importance of integrated models of cell to cell interactions. In this case,
abnormal function of thyroid follicular cells in affected goats may have important

implications for the development and function of oligodendrocytes.

17

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26

Fig. 1 Summary of known (complete lines) and possible (dotted lines)
connections between the genetic defect in B-mannosidosis and decreased
myelination. Capital letters indicate features of B-mannosidosis that have

been confirmed experimentally.

27

Toxic
intermediate
a O
I ’ I
, ’ i
DECREASED ,’ I
s-MANNOSIDASE‘ , ' i‘
ACfiflflTY
I’\ “
I OLIGOSACCHARIDE ‘
,’ ACCUMULATION .l
‘ ’ v T .\ .
Thyroid Hormone" ‘ ~ ‘ ‘ DECREASED
Axis ~ \ ‘. OUGODENDROCYTE
Alteration(s) ~ - - _ s ‘ I NUMBER
. - - - - ~ ‘ \ ¢ " \
GENETIC ” * Oligodendrocyte I ’ DECREASED
DEFECT ‘\ \ a Alterations ' \ ‘ MYEUNATION
\ ’ a ’ \ ’ I
‘\\ ’,v” ’Altered .’
. ‘ s u .. v ’ Oligodendrocyte
Other . , v ’
Defect(s)

Function

Regional Central Nervous System Oligosaccharide Storage

in Caprine B-Mannosidosis

28

29
ABSTRACT

Goats affected with B-mannosidosis, an autosomal recessive disease of
glycoprotein metabolism, have deficient activity of the lysosomal enzyme 8-
mannosidase along with tisSue storage of oligosaccharides, including a
trisaccharide (mannosle1-4N-acetylglucosaminy181-4N-acety1glucosamine) and
a disaccharide (mannoslel-4N-acetylglucosamine). CNS myelin deficiency, with
regional variation in severity, is a major pathological characteristic of affected
goats. This study was designed to investigate regional CNS differences in
oligosaccharide accumulation to assess the extent of correlation between
oligosaccharide accumulation and severity of myelin deficits. The concentrations
of accumulated disaccharide and trisaccharide and the activity of B-mannosidase
were determined in cerebral hemisphere gray and white matter and in spinal
cord from three affected and two control neonatal goats. In affected goats,
content of trisaccharide and disaccharide in spinal cord (moderate myelin
deficiency) was similar to or greater than content in cerebral hemispheres (severe
myelin deficiency). Thus, greater oligosaccharide accumulation Was not associated
with more severe myelin deficiency. Regional B-mannosidase activity levels in
control goats were consistent with the affected goat oligosaccharide accumulation
pattern. The similarity of trisaccharide and disaccharide content in cerebral
hemisphere gray and white matter suggested that lysosomal storage vacuoles,
more numerous in gray matter, may not be the only location-of stored CNS
oligosaccharides.

Key words: lysosome, myelination, storage disease, goat

Running title: OLIGOSACCHARIDE STORAGE IN B-MANNOSIDOSIS

30

In caprine B-mannosidosis, an autosomal recessive disease of glycoprotein
metabolism (Fisher et al., 1986), deficient activity of the lysosomal enzyme 8-
mannosidase (EC 3.2.1.25) is associated with tissue and body fluid accumulation
of a large amount of the enzyme’s putative substrates, including a trisaccharide
(TS, mannosle1-4N-acetylglucosaminy181-4N-acetylglucosamine), a smaller
amount of a disaccharide (DS, mannosle1-4N-acetylglucosamine), and much
smaller quantities of more complex oligosaccharides (Jones and Laine, 1981;
Matsuura et al., 1981; Jones et al., 1984; Matsuura and Jones, 1985; Dahl et al.,
1986; Frei et al., 1986; Hancock et al., 1986). Clinical and pathological
abnormalities in affected goats include facial dysmorphism; neurological signs
including deafness, ataxia, and tremor; cytoplasmic vacuolation; and CNS
hypomyelination (Jones et al., 1983; Kumar et al., 1986). The genetic abnormality
which leads to deficient enzyme activity in goats is currently under investigation.
It is likely that the genetic defect is restricted to the B-mannosidase locus. If so,
the pathogenesis of abnormalities present in B-mannosidosis would be directly or
indirectly linked to deficient activity of B-mannosidase. At the cellular level, toxic
or inhibitory effects of stored, uncatabolized substrate or perturbed glycoprotein
metabolism are possible contributing factors.

A caudal-to-rostral pattern of increasingly severe CNS myelin deficiency
is a major pathological feature of caprine B-mannosidosis. In affected goats, the
spinal cord, which is myelinated relatively early in development, has a moderate
degree of myelin deficiency while the cerebral hemispheres (CH), which are

myelinated later in development, have a severe deficiency of myelin. This pattern

31
and other evidence (Lovell and Jones, 1983; Lovell and Boyer, 1987) suggests
disruption of the myelination process during development. Regional variations
in dysmyelination could be due to accumulation of different amounts of a
substance that impairs oligodendrocyte proliferation, differentiation, or function.

Clinical manifestations of B-mannosidosis are less severe in humans than.
in goats and myelin deficiency has not been detected in human B-mannosidosis
(C00per et al., 1986; Wenger et al., 1986; Dorland et al., 1988). Of potential
importance, DS is the only oligosaccharide detected in the urine of human 8-
mannosidosis patients (Wenger et al., 1986; C00per et al., 1988; Dorland et al.,
1988), sharply contrasting with the TS storage predominance in caprine 8-
mannosidosis. Differences between human and goat oligosaccharide storage
suggest that an accumulation of TS or more complex oligosaccharides could play
a role in the pathogenesis of dysmyelination in caprine B-mannosidosis.

The major objectives of the study were to (1) determine the content of
both TS and DS and the TS:DS ratio in CH gray and white matter and in spinal
cord; (2) assess a possible association of oligosaCcharide content with the severity
of myelin deficiency in these regions; and (3) measure control animal 8-
mannosidase activity in these regions to correlate with the extent of affected
animal TS and DS accumulation. Definition of the regional distribution of 8-
mannosidase substrate accumulation in the CNS and determination of the role
of oligosaccharide accumulation in dysmyelination in caprine B-mannosidosis

were expected outcomes of this investigation.

32
MATERIALS AND METHODS

Materials

T-61 was purchased from American Hoechst (Somerville, NJ). Bio-Gel P-
2, 400 mesh, was purchased from Bio-Rad (Richmond, CA). Redi-Plate silica gel
G thin layer chromatography (TLC) plates and 1-butanol, ACS certified grade,
were purchased from Fisher Scientific (Pittsburgh, PA). Leupetin and pepstatin
A were purchased from Boehringer Mannheim (Indianapolis, IN). All other
chemicals were of reagent grade and purchased from Sigma (St. Louis, MO).
Tissue acquisition

Following euthanasia with T-61, brain and spinal cord from two control
and three affected 2- to 3-day-old animals were removed, sliced, and frozen. For
each animal, 2.2 g samples were excised from frozen spinal cord, CH white
matter, and CH gray matter. CH white matter excision was limited to centrum
semiovale. ‘
Oligosaccharide isolation and analysis

Wagon. Using modifications of methods previously
described (Matsuura and Jones, 1985), tissue samples were minced, brought to
a total volume of 5 ml with triple deionized water, and sonicated for four
minutes using a Heat Systems Ultrasonics model W185 sonifier at a power setting
of 3. Two 0.23 ml samples, containing 0.1 g of tissue each, were withdrawn and
frozen for later B-mannosidase and protein assays. The remaining sonicate was
centrifuged and the supernatant was removed. The pellet was twice dispersed in

water, sonicated, and centrifuged. Supernatants were combined, made 15% in

33

acetic acid by the addition of glacial acetic acid, and after 30 minutes centrifuged
at 12,000g for 20 min. The pellet was twice resuspended in 0.5 ml water and
centrifuged. Supernatants were combined and lyophilized.

W. Lyophilized material was solubilized in 1.0
ml water, applied to a 1.0 X 100 cm, 400 mesh Bio-Gel P-2 column, and eluted.
with water. Fractions of 1.0 ml were collected.

Qligosagcharide analysis. The hexose content in a 200 111 sample from each
fraction was determined by the phenol-sulfuric acid colorimetric method (DuBois
et al., 1956). Thin layer chromatography (TLC) was used to determine the
identity of oligosaccharides contained within the fractions. Samples (40 ul) from
each fraction, along with oligosaccharide standards ((a) glucose oligomer from
acid-digested dextran and (b) a mixture of authentic oligosaccharide samples
purified from affected goat thyroid, see Fig. 1), were applied to silica gel G TLC
plates, and developed using a butanol, acetic acid, water mixture (323:2, by
volume). Hexose was visualized with orcinol-sulfuric acid reagent and heating at
100°C, as previously described (Matsuura and Jones, 1985).

B-Mannosidase assay

Enzymatic activity of B-mannosidase was determined, as previously
described (Jones et al., 1984), except enzyme extraction was in water containing
0.01 M sodium citrate, 0.05 M sodium chloride, 10 mM calcium chloride, 10 mM
manganese chloride, 0.02% (wt/vol) sodium azide, 5% glycerol (vol/vol), 0.2
mg/l leupetin, and 0.7 mg/l pepstatin A instead of plain water. Assays were run

in duplicate, at pH 5.5, and 37°C from 25 ul samples of sonicates of each tissue

34

sample, using 4-methylumbelliferyl-B-D-mannopyranoside as substrate.
Fluorescence levels were assessed using a Gilford FluoroIV fluorimeter. Values
were expressed as nmol 4-methylumbelliferyl released per h per mg protein.
Protein assay

Protein content of both the crude tissue preparations from the,
oligosaccharide isolation samples and the supernatants of B-mannosidase assay
samples were determined, in triplicate, by the Bradford method (Bradford, 1976)
using bovine serum albumin as a standard. Optical absorbance of samples and
dilutions of the standard were analyzed using a Beckman DU-64

spectrophotometer.

RESULTS

Oligosaccharide isolation, quantitation, and characterization

Characterization of crude tissue extracts by thin layer chromatography
(Fig. 1) identified major accumulations of monosaccharide, DS, and TS in
affected animal samples (lane 4) while control animal samples contained
monosaccharide but neither TS nor DS (lane 3). The major oligosaccharides
separated by Bio-Gel P-2 chromatography from affected goat samples included
DS (lane 5) and TS (lane 6). As determined by phenol-sulfuric acid assay,
accumulation of TS and DS varied somewhat among animals (Fig. 2). However,
in each animal, TS content was consistently higher in spinal cord than in either
CH white or gray matter. DS levels were higher in spinal cord than in either CH

region in two of three animals. Mean spinal cord TS and DS levels were each

35

approximately 1.4 times higher than mean levels in either CH region. TS and DS
levels in the affected animals were not substantially different between CH white
matter and gray matter. The TS:DS ratios were not consistently different among
regions. In control animals, TS and DS were not detected in either cerebral
hemisphere or spinal cord tissue.
Regional B-mannosidase activity levels

For both control animals, B-mannosidase activity levels in spinal cord were
higher than in either CH region. Within the cerebral hemispheres, gray and white
matter activity levels were not appreciably different (Fig. 3). B-Mannosidase

activity was not detectable in affected animal samples.

DISCUSSION

These results are the first report of regional CNS oligosaccharide
accumulation and B-mannosidase activity levels in caprine B-mannosidosis. Given
the approximately 2 fold higher level of B-mannosidase activity in control goat
spinal cord compared to CH white or gray matter, the finding of generally higher
levels of oligosaccharide storage in affected goat spinal cord than in CH white
or gray matter is not surprising. Previous studies of thyroid, kidney, and CH gray
matter also have suggested that the level of B-mannosidase activity in control
animal tissues is correlated with the accumulation of oligosaccharides in affected
animal tissues (Jones and Dawson, 1981; Jones and Laine, 1981; Matsuura et al.,
1981; Jones et al., 1984; Pearce et al., 1987). However, the functional

significance of higher B-mannosidase activity in the spinal cord than in CH white

36
or gray matter is not clear.

No determination was made of the molecular structures of the DS and TS
isolated in this study. However, given the conserved nature of the core sugar unit
in N-linked glchproteins (Schachter, 1984), the molecular structure of CNS TS
and DS is likely to be the same as, and can be inferred from, previous structural.
determinations in CH gray matter (Jones and Laine, 1981), peripheral nerve
(Frei et al., 1984), kidney (Matsuura and Jones, 1985), and thyroid (Frei et al.,
1986).

A major goal of this research was to assess in specific CNS regions
whether an association existed between oligosaccharide content and severity of
myelin deficiency. In affected goats, the average content of both TS and DS was
40% higher in spinal cord, an area of moderate myelin deficiency, than in CH,
an area of severe myelin deficiency. Although the number of samples was too low
for meaningful statistical analysis, the consistently higher spinal cord levels of TS
suggest that the etiology of dysmyelination in caprine B-mannosidosis is not
related to accumulation of oligosaccharides. The accumulation of oligosaccharides
in the spinal cord white matter was not separately measured; however, results for
CH gray and white matter were similar, and thus appreciable spinal cord gray
and white matter differences are unlikely. The current research does not rule out
the possibility of inhibitory or toxic effects of complex oligosaccharides, which
have been difficult to study because of their very low tissue concentrations
(Matsuura and Jones, 1985). However, it is probable that the complex

oligosaccharides accumulate somewhat proportionally to TS and DS. Since

37
affected animal CNS has a relatively low concentration of TS and DS, compared
to visceral tissues, the concentration of complex oligosaccharides in CNS is
expected to be low also, and to be distributed similarly to TS and DS
distribution. The existence, in affected goat CNS, of a toxic compound generated
by an alternate metabolic pathway, analogous to galactosylsphingosine in the.
twitcher mouse (Igisu and Suzuki, 1984), remains a possible etiologic mechanism.

A potential limitation in the present study was its focus on postnatal
animals. Myelination in goats is nearly complete at birth, and so tissues at the
"end-stage" of disease were studied. A prenatal developmental study would be
needed to assess oligosaccharide accumulation during active myelin formation to
completely rule out developmentally significant regional differences. However,
although regional differences may exist temporally, it seems likely that, in each
region, the accumulation of oligosaccharides would increase according to that
region’s developmental schedule, and the maximum accumulation would be
reflected in the values measured after birth.

The similarity in storage levels of TS and DS in affected goat CH gray
and white matter and the higher content in spinal cord were unanticipated
results. Morphologically, a greater degree of lysosomal storage vacuolation is
present in CH gray matter cells (predominantly in neurons) than in CH white
matter or spinal cord cells (Jones et al., 1983; Lovell and Jones, 1983; Lovell and
Jones, 1985). It was expected, given the distribution of vacuolation, that CH gray
matter oligosaccharide storage would be greater than CH white matter and spinal

cord storage. The current findings, however, suggest that considerable extra-

38
lysosomal oligosaccharide storage, i.e., cytoplasmic and / or extracellular, is present
in affected goat CNS tissues. This distribution may be unique to the CNS, since
the level of oligosaccharide storage in thyroid, kidney, and liver is roughly
proportional to the extent of cytoplasmic vacuolation in these organs (Jones et
al., 1983; Jones et al., 1984; Frei et al., 1986).

In summary, the data indicate that the extent of regional CNS
accumulation of TS and DS is not associated with regional differences in severity
of myelin deficiency in caprine B-mannosidosis. Although these results do not
directly assess complex oligosaccharide accumulation, it is nonetheless unlikely
that the etiology of dysmyelination in caprine B-mannosidosis is related to
accumulation of oligosaccharides. Thus the differences in oligosaccharide storage
between caprine and human B-mannosidosis do not explain the species

differences with respect to myelination.

Acknowledgments: I wish to thank Drs. Kathryn Lovell and Margaret
Jones for helping design this project and for careful proofreading of the
manuscript. I also wish to acknowledge the work of Dr. Eileen Rathke and Nancy
Truscott in all aspects of this project. I also wish to thank Drs. Karen Friderici,
Kevin Cavanagh, and Rachel Fisher, and Christine Traviss, for helpful
consultation and to acknowledge the technical assistance of Jayne Kelley, Ralph
Common, and Jane Benke with various aspects of this research. This research

was supported by NIH grants NS 20254 to KLL and NS 16886 to MZJ.

39
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DuBois, M., Gilles, K.A., Hamilton, J.K., Rebers, RA, and Smith, F. (1956)
Colorimetric method for determination of sugars and related substances.
Anal. Chem. 28, 351-356.

Fisher, R.A., Rathke, E.J.S., Kelley, J.A., Dunstan, R.W., Cavanagh, K., and
Jones, M2. (1986) Inheritance of B-mannosidosis in goats. Anim. Genet.

17, 183-190.

40

Frei, J.I., Matsuura, F., and Jones, M.Z. (1984) B-mannosidosis: accumulated
peripheral nerve oligosaccharides. Fed. Proc. 43, 1553 (abstract).

Frei, J .I., Matsuura, F., Rathke, EJ.S., and Jones, M.Z. (1986) B-mannosidosis:
Thyroid oligosaccharides. Fed. Proc. 45, 1815 (abstract).

Hancock, L.W., Jones, M.Z., and Dawson, G. (1986) Glycoprotein metabolism.
in normal and B-mannosidase-deficient cultured goat skin fibroblasts.
Biochem. J. 234, 175-183.

Igisu, H. and Suzuki, K. (1984) Progressive accumulation of toxic metabolite in
a genetic leukodystrophy. Science 224, 753-755.

Jones, M.Z. and Dawson, G. (1981) Caprine 8-mannosidosis: inherited
deficiency of B-mannosidase. J. BiOl. Chem. 256, 5185-5188.

Jones, M.Z. and Laine, RA. (1981) Caprine oligosaccharide storage disease:
accumulation of B-mannosyl (1-4) B-N-acetylglucosaminyl (1-4)
B-N-acetylglucosamine in brain. J. Biol. Chem. 256, 5181-5184.

Jones, M.Z., Cunningham, J.G., Dade, A.W., Alessi, D.M., Mostosky, U.V.,
Vorro, J.R., Benitez, J.T., and Lovell, KL. (1983) Caprine
B-mannosidosis: clinical and pathological features. J. Neuropathol. Exp.
Neurol. 42, 268-285.

Jones, M.Z., Rathke, EJ.S., Cavanagh, K., and Hancock, L.W. (1984)
B-mannosidosis: prenatal biochemical and morphological characteristics.
J. Inher. Metab. Dis. 7, 80-85.

Kumar, K., Jones, M.Z., Chmningham, J .G., Kelley, J .A., and Lovell, KL. (1986)

Caprine B-mannosidosisz phenotypic features. Vet. Rec. 118, 325-327.

41

Lovell, KL. and Boyer, PJ. (1987) Dysmyelinogenesis in caprine
B-mannosidosis: ultrastructural and morphometric studies in fetal optic
nerve. Int. J. Dev. Neurosci. 5, 243-253.

Lovell, KL. and Jones, M.Z. (1983) Distribution of central nervous system
lesions in B-mannosidosis. Acta Neuropathol. 62, 121-126.

Lovell, KL. and Jones, M.Z. (1985) Axonal and - myelin lesions in
B-mannosidosis: ultrastructural characteristics. Acta Neuropathol. 65,
293-299.

Matsuura, F. and Jones, M.Z. (1985) Structural characterization of novel
complex oligosaccharides accumulated in the caprine B-mannosidosis
kidney. J. Biol. Chem. 260, 15239-15245.

Matsuura, F., Laine, RA, and Jones, M.Z. (1981) Oligosaccharides accumulated
in the kidney of a goat with B-mannosidosis: mass spectrometry of intact
permethylated derivatives. Arch. Biochem. Biophys. 211, 485-493.

Pearce, R.D., Callahan, J.W., Little, P.B., Armstrong, D.T., Kiehm, D., and
Clarke, J .T.R. (1987) Properties and prenatal ontogeny of B-mannosidase
in selected goat tissues. Biochem. J. 243, 603-609.

Schachter, H. (1984) Glycoproteins: their structure, biosynthesis and possible
clinical implications. Clin. Biochem. 17, 3-14.

Wenger, D.A., Sujansky, E., Fennessey, P.V., and Thompson, J .N. (1986) Human

. B-mannosidase deficiency. N. Engl. J. Med. 315, 1201-1205.

42

FIG. 1. Thin layer chromatography of oligosaccharide standards, CH white matter
crude tissue extracts, and Bio-Gel P-2 isolated oligosaccharides. Samples were
applied to silica gel G TLC plates; plates were developed in butanol, acetic acid,
and water (3:322, by volume); and hexose was visualized with orcinol-sulfuric acid
reagent. 1, glucose oligomer standards; 2, authentic standards: from top,
monosaccharide, DS, TS, pentasaccharide; 3, crude extract of control animal
white matter; 4, crude extract of affected animal white matter; 5, purified DS
from affected animal white matter; 6, purified TS from affected animal white

matter.

43

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m6 . .......... .iw .. ..
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44

FIG. 2. Regional CNS DS and TS content in affected animals. Samples (2 g)
from CH white matter and gray matter and from spinal cord (combined white
and gray matter) of 3 affected (A1, A2, and A3) and 2 control animals were
sonicated with water, and oligosaccharides in water extracts were separated by.
Bio-Gel P-2 column chromatography. Oligosaccharides within fractions were
identified by thin layer chromatography mobility with respect to standards (see
Fig. 1) and the hexose content of each fraction was measured by phenol-sulfuric
acid assay. Additionally, protein content of sonicates was determined (see
methods). In control animals, DS and TS were undetectable. Bars represent, for

affected animal regions, DS or TS content expressed per mg protein.

FIG. 3. Regional CNS B-mannosidase activity in control animals. Samples (0.1
g) from CH white matter and gray matter and from spinal cord (combined white
and gray matter) of 3 affected and 2 control (C1 and C2) animals were sonicated
with an extraction buffer (see methods) and centrifuged. B-mannosidase activity
in each region was determined in supernatant samples by fluorometric
quantitation of the amount of 4-methylumbelliferyl (4-MU) released from 4-MU-
B-D-mannopyranoside added to the samples. Additionally, protein content of
supernatants was determined (see methods). In affected animals, enzyme activity
was undetectable in all regions. Bars represent, for control animal regions, means

of duplicate B-mannosidase activity measurements expressed per mg protein.

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Investigation of Dysmyelinogenesis in Caprine B—Mannosidosic

In Vigo Characterization of Oligodendrocyta

46

47
ABSTRACT

Central nervous system myelin deficiency is a consistent feature of caprine
B-mannosidosis, an autosomal recessive neurovisceral lysosomal storage disease.
To investigate the possibility of an intrinsic oligodendrocyte defect in B-
mannosidosis, oligodendrocyte-enriched glial cultures from the cerebral
hemisphere white matter of 2 affected and 6 control goats were compared with
respect to culture yield and morphology. Fewer oligodendrocytes were cultured
per gram of white matter from affected animals than from control animals.
Galactocerebroside-positive oligodendrocytes from all animals were similar
morphologically at all stages of culture by phase contrast and fluorescence
microscopy. These findings are consistent with in ,2ng morphological observations
and suggest that differentiated oligodendrocytes from affected animals do not
show morphological abnormalities in culture. However, increased numbers of
galactocerebroside-negative bipolar cells, which may be glial progenitor cells,
were present in affected animal cultures. Their presence in increased numbers
in affected animals suggests the possibility of a defect in differentiation to mature
oligodendrocytes, with persistence of the undifferentiated glia during late stages

of development.

48
INTRODUCTION

Myelin deficiency, with regional variation in severity, is a major
pathological feature of the lysosomal storage disease caprine B-mannosidosis. In
a caudal to rostral pattern of increasingly pronounced myelin deficiency, affected
goat spinal cord white matter tracts have moderate myelin deficiency, optic nerve .
and brainstem regions have an intermediate degree of myelin deficiency, and
cerebral hemisphere white matter has severe myelin deficiency (Lovell and Jones,
1983). In Optic nerve from an affected-control pair of 124/ 150 days gestation
fetuses, a 40% reduction in the number of oligodendrocytes was present in the
affected animal (Lovell and Boyer, 1987). This finding, in combination with
qualitative observations in other studies (L0vell and Jones, 1983; 1985), suggested
that oligodendrocyte number is reduced in regions where myelin deficiency is
present. The possibility that affected animal oligodendrocytes produce fewer
myelin sheath segments than control animal oligodendrocytes has been suggested
but not definitively addressed. Other in ziyo observations include: a greater
proportion of oligodendrocytes with vacuoles and dark cytoplasm in affected
goats, beginning prior to development in corpus callosum (Lovell, in
preparation); no ultrastructural difference between affected and control animal
CNS myelin sheaths; and absence of either oligodendrocyte or myelin
degeneration (Jones et al., 1983; Lovell and Jones, 1983; 1985). Taken together,
these. findings suggest that some alteration in oligodendrocyte proliferation,
differentiation, and/or function is present in animals affected with 8-

mannosidosis.

49
Culturing of oligodendrocytes from the CNS of goats affected with 8-

mannosidosis is important to (1) determine if in v_nI_o and in \_/it_ro findings are
consistent, and (2) define any abnormalities that might indicate that an intrinsic
oligodendrocyte defect is present in 8-mannosidosis. Thus, the present study was
designed to characterize affected and control goat oligodendrocytes in 3:ng with
respect to yield per gram white matter and morphological characteristics.
METHODS AND MATERIALS

Materials

Animals studied included six newborn control goats and two newborn
affected goats. Following euthanasia with T-61 (American Hoechst, Somerville,
NJ), the brain was removed and either one or both cerebral hemispheres were
placed in sterile Hanks’ balanced salt solution without calcium and magnesium
containing 10 ug/ml gentamicin and 0.24 ug/ml Fungizone (HBSS-GF), and
transported to tissue culture facilities. Tissue culture procedures were begun
within 30 to 60 minutes of animal death. Unless otherwise noted, reagents were
obtained from GIBCO.
Cell Culture Methods

CNS mixed cell cultures were prepared with modifications of the Percoll
gradient methods described by Hirayama et a1. (1983) and Kim et al. (1983).
Cerebral hemispheres were rinsed 5-7 times with HBSS-GF and cut coronally
into .six sections. White matter was carved from each section with a scalpel,
minced with two single-edged razor blades held in tandem, weighed, and

incubated in HBSS-GF containing 2.5% trypsin and 10 ug/ml DNAse (Sigma)

50
for 30 min at 37° C in an Orbit shaker bath (Lab-Line). After cooling in an ice

bath for 3-5 min, the solution was spun at 750 g for 4 min in a swinging bucket
centrifuge (International Equipment Company) and the supernatant was poured
off. Minimal essential medium (MEM) was added to the pellet and the tissue
was agitated by trituration, passed through 210 um and 50 um mesh Nitex screens.
(Tetco), and diluted to a maximum volume of 40 ml. In each of two
polycarbonate centrifuge tubes, 20 ml of the screened solution was combined with
9 ml Percoll (Sigma) and 1 ml 10X HBSS-GF, mixed thoroughly, then spun at
31,000 g for 30 min at 10°C in a Beckman J2-21 centrifuge in a J17 25° fixed
angle rotor. From each centrifuge tube, the maximal quantity of the clear
”oligodendrocyte-rich" area of the resulting gradient, between the myelin layer on
top and the "cloudy" and red blood cell layers near the bottom (Hirayama et al.,
1983), was withdrawn, diluted to a total volume of 50 ml in MEM, mixed
thoroughly, and spun at 1000 g for 5 min in a swinging bucket centrifuge. The
supernatant was poured off and the pellet was resuspended in 5 ml MEM,
triturated, diluted to 50 ml with MEM, and spun at 750 g for 5 min. The
supernatant was poured off and the pellet was suspended in media. A sample of
the cell suspension was taken for counting by hemocytometer in all 6 control
animal cultures and in 1 of 2 affected animal cultures. The remaining suspension
was placed into Petri plates, and diluted further with medium. Medium consisted
of MEM with D-valine (GIBCO # 320-2570), 10% heat-treated fetal calf serum
(Hyclone Laboratories), an additional 6 mg% glucose, 0.12 ug/ml gentamicin,

and 0.12 ug/ml Fungizone. Cells were incubated in an humidified chamber with

51

5% CO1 at 37°C. On day 3, medium, containing floating clumps of cells, was
removed from Petri plates, triturated, and spun at 1,000 g for 10 min. The cell
pellets were resuspended in media, plated on 15-mm-diameter glass coverslips
(Bellco) precoated with 1 mg/ ml poly-L-lysine (Sigma), and incubated in Petri
plates. Medium was added to Petri plates after 12-16 hours. Subsequently, cells.
were fed every 3-4 days by replacement of half of the total medium volume with
fresh medium.
Immunolabelling

For immunofluorescence labeling of oligodendrocytes, coverslips were
rinsed in fresh MEM, then incubated for 15 min with monoclonal anti-
galactocerebroside (anti-GC, provided by'Dr. Joyce Benjamins) diluted 1:50 in
MEM. Coverslips were rinsed in MEM, then incubated for 30 min with
fluorescein-conjugated goat anti-mouse IgG (ICN Biochemicals) diluted 1:20 in
MEM. Coverslips were rinsed in MEM, cells were fixed in 4% paraformaldehyde
for 10-15 min., and coverslips were rinsed with 0.05 M Tris and mounted as
below. For immunofluorescence labeling of astrocytes, cells were rinsed in MEM,
fixed first in 4% paraformaldehyde for 10 min and then in -20°C methanol for
20 min. Cells were labeled for 60 min with a prediluted monoclonal anti-GFAP
cocktail (Biomedical Technologies), then, after rinsing in Tris, incubated for 30
min with FITC-conjugated goat anti-mouse antibody (ICN Biochemicals) diluted
1:20in Tris and then rinsed with Tris. For staining controls, normal mouse serum
(Dako), diluted 1:50, and prediluted myeloma protein control solution

(Biomedical Technologies) replaced primary antibody. Coverslips were mounted

52

onto glass slides with Aquamount (Lerner Laboratories). All procedures, with the
exception of methanol fixation, were carried out at room temperature.
Microscopy

Cells were examined and photographed at various stages of culture using
a Nikon TMS inverted microscope. Immunofluorescence labelled cells were.
examined and photographed using a Leitz Ortholux II fluorescence microscope
equipped with phase contrast optics. Morphological assessment of cultured cells
included examination by phase contrast and fluorescence microscopy over a
period of 0 (at plating) through 30 days on coverslips (DOC). Phase contrast
morphological assessment included examination of cell body and processes and
assessment of changes during culture. Additionally, immunolabelled cells were
examined by (1) fluorescence then phase contrast, and (2) phase contrast then
fluorescence to (a) assess the approximate proportion of GC+ cells among plated
cells; (b) assess GC-immunofluorescence pattern of oligodendrocytes; and (c)
check for GC+ staining in cells with phase contrast oligodendrocyte-like

morphology.

RESULTS
Cells from Control Animals
Control animal cell yield from the "oligodendrocyte-rich" layer of the
Percoll gradient ranged from 0.86 X 10‘ to 3.25 X 10‘ cells per gram white
matter. Cell viability, assessed by trypan blue exclusion, was consistently greater

than 93%. In all cultures, after 3 days in Petri plates, numerous clumps of cells

53

were seen floating in the media while other cells had attached and typically had
a flat appearance. The vast majority of clumped cells were uniform in appearance
(Fig. 1 A). Most were round, had a phase bright halo, and had a yellow-orange
hue. Even after vigorous trituration, many floating cells spun down from medium
removed from the Petri. plates remained in clumps at plating on poly-lysine.
coated coverslips, although some individual cells were present also. Within 2-5
days on coverslips (DOC) most control animal cells had established processes.
Processes were initially short, extending about 2-5 um and often curving back
toward the cell body. Cell body diameters were typically 8 to 15 um. When
examined between 3-5 DOC, over 70% of plated cells were GC+ (Fig. 2 A,B).
Between 7 and 30 DOC, oligodendrocyte morphology was similar to that
reported in oligodendrocyte cultures isolated from the brains of mature sheep
(Yim et al., 1986), cows (Lisak et al., 1981), rats (Hirayama et al., 1983), and
humans (Kim, 1983). The number, extent of branching, length, and diameter of
processes varied among oligodendrocytes. Most oligodendrocytes established
relatively simple, GC+ processes (Fig. 3 A), although some deve10ped elaborate,
fine branches off of main processes. Some cells elaborated GC+ membranous
sheets from their processes or from their cell bodies (arrow, Fig. 3 D).
Occasional oligodendrocytes developed long, thick, GC+ processes which
extended as far as 50 um with node-like areas periodically along their shafts.
Most oligodendrocytes remained in direct contact with Coverslips. Using both
phase contrast and fluorescence microscopy, all cells with phase contrast

oligodendrocyte-like appearance were found to be GC+ , and few GC- cells were

54

seen early in control animal cultures. Occasional small, round, bipolar, GC- cells
with 48 um cell body diameter were present (arrows, Fig. 4 A). These cells
plated out exclusively on top of flat cells which had the appearance of astrocytes.
By 10—14 DOC, a virtual monolayer of flat, predominantly GFAP-i- cells was
present on coverslips which made phase contrast observations of oligodendrocytes .
difficult.
Cells from Affected Animals

Affected animal cell yield from the "oligodendrocyte-rich" layer of the
Percoll gradient from one animal was 0.29 X 10‘ cells per gram white matter.
Cell viability, assessed by trypan blue exclusion in one animal, was 94%. As in
control animal cells, floating clumps of cells were noted in the medium during
the Petri plate step. However, while some clumped cells from affected animals
were similar in size and phase contrast appearance to control cells, many cells
were small and phase dark (arrowhead, Fig. 1 B). After plating of cells spun
down from Petri plate media, only a sparse population of oligodendrocytes was
identified by immunofluorescence. Only 10% to 20% of plated affected goat
clumped cells were GC+ at 3 DOC (Fig. 2 C,D). Affected animal
oligodendrocyte appearance, with respect to process and sheet formation, was
similar to that of control animal oligodendrocytes by both phase contrast and
immunofluorescence parameters at all stages of culture (Fig. 3 B,C,E). GC-
labelling characteristics of affected animal oligodendrocytes, including pattern of
fluorescence and qualitative intensity of fluorescence, were similar to control

animal oligodendrocyte characteristics. Using both phase contrast and

55

fluorescence microscopy, all cells that had phase contrast oligodendrocyte-like
appearance were GC+. Additionally, bipolar, GC- cells (arrows, Fig. 4 B), were
present in much greater numbers in affected animal cultures. Cell body diameter
of bipolar cells was between 4 and 8 um. The eventual development of a
monolayer of flat, GFAP+ cells was similar to the control culture pattern-
Neither cytoplasmic vacuolation nor evidence of oligodendrocyte degeneration

or death was noted in affected or control cultures.

DISCUSSION

This is the first report of the culture of oligodendrocytes from animals
affected with B-mannosidosis. An important result of this study is the observation
that affected and control animal oligodendrocytes are morphologically very
similar in xigo. Similarities are present with respect to (1) appearance of
processes and cell bodies, (2) appearance of sheet-like membranous proliferation
from a similar proportion of cells, and (3) pattern of immunofluorescence on
cells identified by phase contrast as oligodendrocytes. These results are consistent
with in y1_'_v_o morphological observations, i.e., affected animal oligodendrocytes
were reduced in number but produced normal-appearing myelin sheaths (Lovell
and Boyer, 1987; Lovell and Jones, 1983; 1985). In vita evaluation of cells by
electron microsc0py will be needed to assess how cultured oligodendrocytes
compare with respect to the in v_iyo finding of an increased proportion of

oligodendrocytes with dark cytOplasm and vacuoles in affected animals (Lovell,

56
in preparation).

Two lines of evidence from this research indicate that significantly fewer
oligodendrocytes were present in affected animal cultures than in control animal
cultures. First, counts of cells isolated from the Percoll gradient from one
affected animal found between 10% and 40% of the number of cells isolated in,
the six control animal cultures. While these counts include some proportion of
"contaminating" cells like astrocytes, they nonetheless reflect the number of cells
present in the "oligodendrocyte-rich region" of the Percoll gradient. Second, a
much lower percentage of affected animal clumped cells were GC+ after plating
on coverslips and there was a greater percentage of GC- bipolar cells. It is
possible that differences in oligodendrOcyte buoyancy, due to cytoplasmic
vacuolation, might lead to failure in isolation of a higher percentage of affected
animal oligodendrocytes. However, a large portion of the Percoll gradient was
harvested, to include a relatively broad band of densities, and loss of some
oligodendrocytes due to buoyancy differences would not explain the lower
percentage of GC+ cells on coverslips. Thus, it is most likely that the decreased
number of oligodendrocytes observed in yigo reflects the decreased number in
mo in areas of affected goat CNS with severe myelin deficiency (Jones et al.,
1983; Lovell and Boyer, 1987; Lovell and Jones, 1983; 1985).

The genetic defect in B-mannosidosis most likely affects the 8-
mannosidase enzyme gene locus. Decreased levels of tissue and plasma activity
of the lysosomal form of this enzyme are found in all affected animals (Jones and

Dawson, 1981; Jones et al., 1984) and produce tissue accumulation of the

57
putative oligosaccharide substrates for the enzyme (Jones and Laine, 1981; Jones
et al., 1984; Matsuura and Jones, 1985; Matsuura et al., 1981). Oligosaccharide
storage is thought to explain the cytoplasmic vacuolation seen in yjyo in most cell
types of affected animals, although cytoplasmic storage may also be present
outside of lysosomal storage vacuoles. While mild to moderate vacuolation is.
seen in v_igo in oligodendrocytes (Jones et al., 1983; Lovell and Boyer, 1987;
Lovell and Jones, 1983; 1985), no cytoplasmic vacuolation is seen by phase
contrast microscopy in cultured oligodendrocytes up to 30 DOC or in astrocytes
up to 90 DOC. Phase contrast microscopy should provide excellent visualization
of vacuoles if they are present. This apparent lack of vacuolation of cultured
affected animal glial cells is consistent with similar observations in affected
animal fibroblast cultures. Fibroblasts contain vacuoles in Elm (Jones et al.,
1983). However, in u‘go, both the presence of oligosaccharides in medium and
in cytoplasm have been demonstrated in fibroblast cultures, in the absence of
cytOplasmic vacuolation (Hancock et al., 1986; and unpublished observations).
The absence of vacuoles in cultured cells could be related to cell isolation
procedures, which cause some disruption of the normal architecture of cells.
Thus, loss of some portion of membrane and cytoplasm, and possibly lysosomal
storage vacuoles, could occur during the isolation. Lack of formation of vacuoles
during the culture period may be related to different behavior of the cells in
culture than in gigo, possibly with (1) different storage locations for uncatabolized
oligosaccharides (e.g. cytoplasmic versus lysosomal), (2) altered metabolism with

less accumulation of oligosaccharides and thus fewer or smaller storage vacuoles,

58
and (3) exocytosis of uncatabolized oligosaccharides into the medium as opposed
to storage.

Although not specifically quantitated, a substantial increase in the
percentage of GC- bipolar cells was present in affected animal cultures compared
to control animal cultures. Bipolar cells had Percoll gradient density.
characteristics within the range of oligodendrocytes and floated and clumped
together, along with oligodendrocytes, during the Petri plate step.
Morphologically similar cells have been identified as glial progenitor cells in
mixed glial cultures from neonatal rat (Raff et al., 1983; Temple and Raff, 1986)
and fetal sheep (Elder et al., 1988), using anti-GC and anti-A,B,, a monoclonal
antibody directed against a complex ganglioside (Eisenbarth et al., 1979). A small
number of bipolar cells was also seen in all control goat cultures, but the ratio
of bipolar cells to oligodendrocytes was clearly low in control cultures. If the
bipolar cells are glial progenitor cells, these results may suggest that an increased
number of glial progenitor cells remain undifferentiated in affected animals.
Since the affected animal cells were isolated from cerebral hemisphere white
matter, where a decreased number of mature oligodendrocytes are found, an
abnormality or block in differentiation of progenitor cells to oligodendrocytes
may be indicated. The possibility remains, however, that affected and control
animal white matter contain comparable numbers of bipolar cells per gram tissue
and that the appearance of an increased proportion of bipolar cells in affected
animal cultures is due to the substantial reduction in number of mature

oligodendrocytes. Additional studies are needed to further investigate bipolar

59
cells and to determine the role of impairment of oligodendrocyte development

in caprine B-mannosidosis.

60

ACKNOWLEDGEMENTS

I wish to acknowledge the guidance and assistance of Dr. Kathryn Lovell
with all aspects of this project. I also wish to thank Drs. Pamela Knapp, Charissa
Dyer, Robert Lisak, Sara Szuchet, and James Trosko, and Dorothy Okazaki, for.
consultation about and assistance with developing cell culture techniques. Dr.
Joyce Benjamins provided anti-GC antiserum. The technical assistance of Ralph
Common, Nancy Truscott, Donna Craft, and Jayne Kelley with various aspects
of this research is gratefully acknowledged. This study was supported by NS-
20254 to K.L.L., NS-16886 to Dr. M.Z. Jones, and RR-05655.

61

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develops in vitro into an astrocyte or an oligodendrocyte depending on
culture medium. Nature 303:390—396.

Temple, 8., and Raff, M.C. (1986) Clonal analysis of oligodendrocyte
development in culture: evidence for a developmental clock that counts
cell divisions. Cell 44:773-779.

Yim, S.H., Szuchet, S., and Polak, RE. (1986) Cultured oligodendrocytes: a role
for cell-substratum interaction in phenotypic expression. J. Biol. Chem.

261:11808-11815.

64

Fig. 1. Control (A) and affected (B) animal cells after 3 days in culture
in Petri plates. Floating cells isolated from both affected and control animal CNS
white matter were clumped together. Control animal cells were relatively uniform
in size and most had a phase bright appearance (arrow). Most affected animal
cells were small and phase dark (arrowhead) although some phase bright cells

were present (arrow). 250X.

Fig. 2. Phase contrast (left) and GC immunofluorescence-labelled (right)
micrographs from identical fields from control (AB) and affected (C,D) animal
cells'3 days after plating of floating cells on poly-L-lysine coated glass coverslips.
Most clumped control animal cells were GC+ (B) while many clumped affected

animal cells (e.g. C, arrows) were GC- (D). 840x.

65

 

 

66

 

Fig. 3. Control (A,E) and affected (B-D,F) animal cells between 12 and
21 days on coverslips labelled by GC immunofluorescence. Although there were
fewer oligodendrocytes present in affected animal cultures, the number and,

length of oligodendrocyte processes in control (A,E) and affected (B-D,F) animal

 

cultures was quite similar. Also, formation of GC+ membranous sheets was 1

similar in control (E, arrow) and affected (F, arrow) cultures. 630x.

Fig. 4. Control (A) and affected (B) animal cells at 8 days on coverslips.
Many bipolar cells (arrows) were present in affected animal cultures while few
were present in control animal cultures. No cyt0plasmic vacuoles were noted by

phase contrast microscopy in control or affected animal cells. 160X.

67

Fig. 3

 

Astrocyte Abnormalities in Caprine B—Mannosidosis:
An Immunocytochemical Analysis of Optic Nerve

68

69
ABSTRACT

Central nervous system abnormalities in caprine B-mannosidosis, an
autosomal recessive disease of glycoprotein catabolism, include myelin deficiency
with regional variation in severity and reduced numbers of oligodendrocytes in
areas of severe myelin deficiency. A previous ultrastructural and.
immunocytochemical study demonstrated an increased density of astrocyte
processes in CNS regions with moderate to severe myelin deficiency. The current
study sought to assess developmental aspects of gliosis through an
immunocytochemical investigation of glial fibrillary acidic protein (GFAP)
reactivity in optic nerves of animals ranging in age from 115/150 days gestation
to 31 days postnatal.

The density of GFAP+ astrocyte processes was quantitatively greater in
affected animal optic nerves than in control animal optic nerves as early as
115/ 150 days gestation. However, astrocyte process density did not increase
substantially with increasing age. Additionally, no increase in the number of
GFAP+ astrocyte cell bodies was present in affected goat optic nerves compared
to control goat optic nerves. These results suggest that astrocyte changes are
present in the affected animal optic nerve early in the process of myelination and
that the extent of change, compared to controls, does not increase substantially
with age. Increased astrocyte process density at a time early in myelination
suggests that gliosis is not secondary to deficient myelination alone and may.
instead take place in response to other abnormalities, possibly in response to a

decreased number or abnormal development of oligodendrocytes. Additionally,

70

the increased density of GFAP+ astrocyte processes appears to be due to a
greater number of processes extending from each astrocyte, although the
possibility of an increased number of astrocytes with redistribution of GFAP from

cell bodies into processes has not been ruled out.

71
INTRODUCTION

A caudal-to-rostral pattern of increasingly severe central nervous system
(CNS) myelin deficiency is a major pathological feature of caprine B-
mannosidosis, an autosomal recessive disease of glycoprotein metabolism (20).
In affected goats, the spinal cord, which is myelinated relatively early in.
development, has a moderate degree of myelin deficiency while the cerebral
hemispheres (CH), which are myelinated later in development, have a severe
deficiency of myelin. This pattern and other evidence suggest that disruption of
the myelination process takes place during development. Both quantitative (19)
and qualitative observations (20,21) have suggested that oligodendrocyte number
is reduced in regions where myelin deficiency is present.

Astrocytes respond to central nervous system (CNS) pathological changes
with “gliosis." Gliosis consists of (1) astrocyte proliferation and/or (2) astrocyte
hypertrophy, with an increased number of extended processes and an increased
expression of the astrocyte-specific intermediate filament glial fibrillary acidic
protein (GFAP) (3,17). Gliosis has been described in a variety of CNS
pathological conditions including trauma, inflammatory diseases, hepatic
encephalopathy, degenerative diseases, and aging (3,7,10,29). Additionally, gliosis
has been identified in the CNS of various myelination mutant mice, including
jimpy, quaking, shiverer, and mld mutants (1).

. A previous immunohistochemical and electron microscopic study in goats
affected with B-mannosidosis demonstrated an increased density of astrocyte

processes without an increased number of cell bodies in CNS regions with

72
moderate to severe myelin deficiency (18). Possible explanations of this gliosis
include reactive response to ( 1) deficient myelin formation, (2) oligodendrocyte
abnormalities, or (3) metabolic perturbations inherent to affected animals.
Additionally, it is possible that a primary abnormality in astrocyte multiplication
in B-mannosidosis could lead to inhibition of myelination. To help clarify some.
of these issues, the current study was designed to assess developmental aspects
of gliosis through an immunocytochemical investigation of GFAP reactivity in
optic nerves of animals ranging in age from 115 / 150 days gestation to 31 days
postnatal. Objectives were to (l) assess whether gliosis was present during early
myelination, (2) determine whether gliosis increased during development, and (3)
describe features of gliosis in the optic nerve for later comparison with features

in other CNS regions.

73
METHODS AND MATERIALS

i s A i ii n H n [in

Age-matched pairs of affected and control animals at 115/ 150 and
124/ 150 days gestation (1 pair at each age), 3-5 days postnatal (2 pairs), and 4
weeks postnatal (1 pair) were used in this study. Animals were classified as,
affected, carrier, or normal by phenotypic characteristics (12,14,16) and plasma
B-mannosidase assay (4,6,8). Both carrier and normal animals were used as
controls. T-61 euthanasia was used for neonatal and older goats. Fetuses,
obtained by cesarean section, were perfused with a 2% paraformaldehyde and
2% glutaraldehyde buffered solution before necropsy. Optic nerve samples from
within 0.5 cm of the orbit were excised and (1) fixed in 4% glutaraldehyde for
2-4 hrs, post-fixed in 1% osmium tetroxide, and embedded in an Epon-Araldite
mixture and (2) fixed in 4% paraformaldehyde for 24 hours and stored in 0.05
M Tris buffer containing 0.02% sodium azide until Vibratome sectioning. Thick
sections (1 um) were cut from Epon blocks, placed on glass slides, and either left
unstained or stained with toluidine blue. Sections of 50 or 60 um were cut with
a series 1000 Vibratome (Technical Products International) and placed in Tris
containing 0.02% sodium azide.
Iamgnooytoohomisgy

Epon immunocytochemistry was carried out using modifications of the
methods. of Trapp et al. (28). Glass slides containing 1 um semithin sections were
heated overnight in a 68°C oven; etched by (a) incubation of slides for 15 min

in sodium ethoxide (saturated sodium hydroxide in absolute ethanol aged for

74

more than 2 weeks, diluted 1:2 with absolute ethanol), (b) a 1 min rinse in each
of three absolute ethanol baths, and (c) incubation in 0.2% sodium hydroxide in
water for 5 min; and rinsed in 0.05 M Tris buffer bath (pH=7.4). All subsequent
rinses were for 15 min in Tris. All incubations were carried out at room
temperature. All antibody dilutions were in Tris containing 1% swine serum-
Blocking serum (Tris containing 3% swine serum (Dako)) was applied to sections
for 60 min, then blotted off. Sections were incubated for 60 min with primary
antibody (1:100); rinsed; incubated for 60 min with secondary antibody (1:40);
rinsed; incubated in peroxidase-anti-peroxidase complex (Dako) (1:80); and
rinsed. For staining control slides, Tris and normal rabbit serum (Dako) (1:100)
were substituted for primary antibody. Peroxidase labeling was revealed by
incubation for 10 min with diaminobenzadine (Sigma), 0.5 mg/ml, in water
containing 0.01% hydrogen peroxide. DAB staining was intensified by 5 min
exposure of sections to 2% osmium tetroxide. Finally, sections were dehydrated,
cleared, and coverslipped using Permount (Fisher Scientific) as a mounting
media.

Vibratome immunochemistry, using methods modified from Koeppen et
al. (15), was carried out in tissue culture wells. Sections were incubated overnight
in Tris containing 1.5% sodium chloride, rinsed, and incubated for 1 h in a
"suppressor serum" consisting of 10% swine serum, 4% bovine serum albumen
(Sigma), and 0.1% Triton X-100 (Sigma) in Tris. Suppressor serum was pipeted
off and sections were incubated in a 1:100 dilution of anti-GFAP, or 1:100 rabbit

serum or Tris for staining controls, each containing 1% swine serum and 0.1%

75
Triton X-100. Remaining solutions and procedures were as described for paraffin
sections, except DAB incubation was for 1-2 min. After mounting on glass slides
precoated with a aqueous mixture of 0.01% chrome alum and 0.1% gelatin,
sections were dehydrated, cleared, and coverslipped.
i i r

GFAP-labelled Epon, paraffin and Vibratome sections were examined and
photographed with a Nikon Microphot-FX photomicroscope. A qualitative
assessment of distribution of GFAP+ reactivity and number of astrocyte cell
bodies was made.
Imago Analysis

The proportion of areas occupied by GFAP reaction product was
quantified using a Nikon-Joyce Loebl Magiscan Ila image analyzer system. Three
GFAP-labelled sections from either 2 or 3 blocks from fetal pairs at 115 / 150 and
124/ 150 days gestation, two pairs of 3-day-old animals, and one pair of 3-week-
old animals were examined. Fifteen consecutive oil immersion fields, occupying
a total of 1.2 X 10‘ um’, were analyzed on each section. Fields were adjusted to

exclude cell bodies, perifascicular connective tissue, and vascular regions.

m

Assessed qualitatively, a substantially greater density of GFAP+ astrocyte
profiles was present in the optic nerve of affected animals compared to control
optic nerves as early as 115 /150 days gestation and as late as four weeks of age

(Fig. 1). At higher magnification (not shown) it was found that affected animal

76
astrocyte process profiles were both more numerous and of larger cross-sectional
area than control animal profiles. As quantitated by image analysis, the
proportion of area covered by GFAP reaction product was consistently greater
in affected animals than in their age-matched controls (Fig. 2). However,
comparing affected animal results, only a modest difference in GFAP reaction.
product density was present between the 115/150-and the 124/ 150 days gestation
fetal optic nerves, and no difference was present among optic nerves of the
124/ 150 days gestation fetus and older animals. Although a greater density of
GFAP+ astrocyte profiles is present in optic nerve of affected animals, no
corresponding increase in the number of GFAP+ cell bodies was present (Fig.

3).

DISCUSSION

The optic nerve was chosen for analysis to (1) represent an area of
intermediate myelin deficiency in affected animals and 'to (2) provide a
morphologically uniform area for comparisons between affected and control
animals and among animals of different ages. Because of the well documented
glial cell and blood vessel variations along the length of the optic nerve (26,27),
this study was restricted to sections of optic nerve nearest the orbit.

A pronounced increase in the proportion of area occupied by GFAP-
reactive astrocyte processes was present in all affected goat optic nerves

examined, and qualitative light microscopic evidence from GFAP-reactivity in the

77

affected animal optic nerve Vibratome sections found that cell body density is
not increased above normal levels. These findings suggest that the astrocyte
process density increase in affected animals is probably secondary to astrocyte
hypertrophy, with an increase in number of processes extended from each
astrocyte, without increased astrocyte number. Although astrocyte proliferation.
is an element of gliosis in some CNS pathological processes (10,17), astroglial
hypertrophy without concomitant proliferation leads to marked gliosis as well
(24,25). Additionally, a study in the myelination mutant jimpy mouse attributed
gliosis to glial cell hypertrophy after ruling out astrocyte proliferation (2).
Nonetheless, identification of astrocyte cell bodies in the current study rests on
the labeling of GFAP. Redistribution of GFAP away from the cell bodies of
astrocytes and into the newly extended processes could leave some proportion of
cell bodies undetected by the immunocytochemical staining procedure employed
here. An earlier electron microscopic and immunocytochemical study suggested
that such a pattern may occur in the cerebral hemisphere white matter of
affected goats (18). This possibility needs further investigation.

A decreased volume of tissue in which the same number of astrocytic
processes are more densely packed could lead to the appearance of increased
density of astrocyte processes. An increased amount of interfascicular connective
tissue and a decrease in fascicle area in the affected animal optic nerve (Lovell,
unpublished observations), so condensation of fascicle contents is a possibility;
However, cross-sectional area in optic nerves from a pair of affected and control

fetuses at 124/ 150 days gestation, during early myelination, was nearly identical

78
(19), and there is no corresponding increase in packing density of GFAP-reactive
cell bodies. Nonetheless, gliosis is present early in myelination, and may well
compensate for decreased tissue density by playing a space-occupying role.

A substantial difference in astrocyte profile density between control and
affected animals was preSent as early as 115/ 150 days gestation, an early point.
in myelination. Similar findings were recently reported in the jimpy mouse, with
gliosis present in various CNS regions, at a point preceding or early in
myelination in the region (2). Increased astrocyte density in the optic nerve of
animals affected with B-mannosidosis at an early stage in myelination suggests
that gliosis is not entirely a reaction to deficient myelination. Other elements
possibly responsible for triggering the gliotic reaction may include abnormalities
in oligodendrocyte differentiation. and maturation or the accumulation of
oligosaccharides.

Animals affected with caprine B-mannosidosis have deficient activity of the
lysosomal enzyme B-mannosidase and an associated tissue and body fluid
accumulation of a large amount of the enzyme’s putative substrates, including a
trisaccharide (mannosle1-4N-acetylglucosaminy181-4N-acetylglucosamine), a .-
smaller amount of a disaccharide (mannosle1-4N-acetylglucosamine), and much
smaller quantities of more complex oligosaccharides (5,9,13,14,22,23). Astrocytes,
like oligodendrocytes, have a variable but generally modest degree of cytoplasmic
vacuolation, with presumed lysosomal accumulation of oligosaccharides (12). A

recent study has suggested that levels of oligosaccharide storage are not

79
associated with myelin deficiency (Boyer et al., in preparation). However, it is
nonetheless possible that astrocytes, which appear to play a key metabolic and
homeostatic role in maintaining the microenvironment of the CNS (3,7,11), could
respond by gliosis to the abnormal presence of oligosaccharides or to an
undefined CNS injury secondary to oligosaccharide presence.

These results suggest that astrocyte changes are present in the affected
animal optic nerve early in myelination and that the extent of change, compared
to controls, does not increase substantially with age. Increased astrocyte process
density early in myelination suggests that astrocytosis may not entirely be' a
reaction to deficient myelination and may take place in response to other
abnormalities, possibly defects in oligodendrocyte development. Additionally, the
increased density of GFAP+ astrocyte processes appears to be due to a greater
number of processes extending from astrocytes, although the possibility of an
increased number of astrocytes with redistribution of GFAP from cell bodies into
processes has not been ruled out. Further work is needed to characterize
astrocyte changes in regions of both severe myelin deficiency, such as the corpus

callosum, and in regions of mild myelin deficiency, such as the spinal cord.

80
Acknowledgments:

I wish to acknowledge the the assistance of Dr. Kathryn Lovell with all
aspects of this project. I also wish to thank Dr. M.Z. Jones for valuable
discussions and for her important contributions to this research, Dr. 1. Duncan
for preliminary trials with and consultation about Epon section.
immunocytochemistry, and Dr. R.P. Skoff for valuable discussions and
consultation on vibratome immunocytochemistry. Cesarian sections were
performed by Dr. N. Kent Ames and anesthesia was administered by Dr Cheryl
Blaze, both from the Michigan State University Department of Large Animal
Clinical Sciences. The excellent technical assistance of Kay Trosko, Nancy
Truscott, Ralph Common, and Jayne Kelly are very much appreciated. This
research was supported by NIH grants NS-20254 to Dr. KL. Lovell and NS-
16886 to Dr. M.Z. Jones.

81

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Best TT, Skoff RP, Bartlett WP: Astroglial plasticity in hemizygous and-
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Cavanagh K, Dunstan RW, Jones MZ: Plasma 0- and B-mannosidase
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Hansen LA, Armstrong DM, Terry RD: An immunohistochemical
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Skoff RP, Toland D, Nast E2 Pattern of myelination and distribution of
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T rapp BD, Itoyama Y, Sternberger NH, Quarles RH, Webster HdeF:
Immunocytochemical localization of P. protein in golgi complex
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Vernadakis A: Changes in astrocytes with aging. In Astrocytes, edited by
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85

Fig. 1. GFAP-labelled, Epon-embedded optic nerve sections from age-matched
control (left) and affected (right) goats at 115/ 150 days gestation (A,B), 3 days
postnatal (CD), and 4 weeks postnatal (E,F). A substantial increase in the
number of GFAP+ astrocyte profiles is present in the affected animal sections.
The difference between affected and control animals is present as early as

115 / 150 days gestation. 790 X.

 

        

   

 

 

    

 

4 4.7.1... «IMF; . ...I...... -.............4 1...

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Density of GFAP Reaction Product

87

 

0.4

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[2:] 2

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Age

Fig. 2. GFAP reaction product density determined by image analysis in age-
matched control and affected animal optic nerves. Density is defined as the ratio
of area occupied by GFAP reaction product to total area. Bars represent staining
density in 1.2x10‘ um2 of optic nerve at each age. Density of the 3-5 day old
animals was averaged (labelled "3 Days"). Affected animals have a greater density
of GFAP reaction product at all ages examined, however, affected animal GFAP

reaction product density does not rise appreciably with age.

Fig. 3. GFAP-labelled 60 um Vibratome sections of optic nerve from 3-day-old
control (A) and affected (B) goats. Although a greater density of GFAP+
astrocyte processes is present in the affected animal, no corresponding increase

in the number of GFAP+ cell bodies is present. 315 X.

89

       
         

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Caprine B-Mannosidosis:

Abnormal Thyroid Structure and Function in a Lysosomal Storage Disease

90

91
ABSTRACT

Deficient activity of the lysosomal enzyme B-mannosidase leads to
widespread tissue accumulation of oligosaccharides in caprine B-mannosidosis,
an autosomal recessive neurovisceral storage disease. Severe thyroid
morphological abnormalities found in a previous light microscopic survey of
tissues from neonatal affected goats suggested the possibility of impairment of
function. Since considerable evidence indicates that thyroid hormones play an
important role in regulation of myelination, deficient thyroid hormone
concentrations, if present, could be a factor in the hypomyelination seen in
affected animals. Thus, this study was designed to characterize thyroid structure
and function in B-mannosidosis.

To investigate developmental aspects of structural abnormalities, thyroids
from six pairs of affected and control animals ranging in age from 96/150 days
gestation to 3 days postnatal were analyzed by light and electron microscopy.
Major findings in affected animal thyroids, as early as 96/ 150 days gestation,
included follicle irregularities and pronounced presence of lysosomal storage
vacuoles in all cell types, particularly in follicular cells. The degree of cytoplasmic
vacuolation increased with advancing age. To assess thyroid function, thyroid
hormone concentrations were determined in six age-matched, neonatal pairs of
affected and control goats. Significantly decreased thyroid hormone
concentrations were present in affected animals. It is hypothesized that reduced
thyroid hormone function plays a role in the pathogenesis of hypomyelination in

affected animals. This study comprises, to our knowledge, both the most

92
complete description of developmental abnormalities and the first report of
abnormal function in an endocrine organ in a lysosomal storage disease. Further,
this report suggests that the pathogenesis of central nervous system lesions could
involve systemic perturbations induced by a genetically determined deficiency of

a lysosomal hydrolase.

93

+

94

INTRODUCTION

The fundamental genetic defect in caprine B-mannosidosis, an autosomal
recessive disease of glchprotein metabolism, is thought to affect the 13-
mannosidase gene. Deficient activity of the lysosomal enzyme B-mannosidase in
affected goats leads to tissue accumulation of related oligosaccharide substrates .
(26,34,35). Previous light microscopic tissue surveys in four affected and two
control neonatal animals showed that some degree of cytoplasmic vacuolation
was present in most parenchymal, vascular, and connective tissue cells in affected
goat tissues, but that thyroid follicular epithelial cells were particularly heavily
vacuolated (25). The extensive degree of vacuolation raised the issue of possible
functional alteration due to morpholOgical abnormalities or to metabolic
perturbations resulting from deficient B-mannosidase activity. Although
endocrine organs are the site of considerable substrate deposition in some
lysosomal storage diseases (22,47), the possibility of a coexisting functional
abnormality has not previously been addressed. This study was designed to
investigate structure-function correlations in a severely affected endocrine organ
in a lysosomal storage disease and to "consider the possibility that the
pathogenesis of hypomyelination in caprine B-mannosidosis is secondary to

abnormal thyroid function.

EXPERIMENTAL DESIGN
Fetal and newborn goat kids were obtained from breedings within two

goat herds: herd A, consisting of normal goats (homozygous unaffected for the

95
genetic defect), and herd B, consisting of carrier goats (heterozygous for the
genetic defect). Experimental animals were classified as affected, carrier, or
normal by phenotypic characteristics (25,27,29) and plasma B-mannosidase assay
(10,14,17). Both carrier and normal animals were used as controls.

To characterize developmental aspects of thyroid structural abnormalities-
in B-mannosidosis, thyroid samples from age-matched pairs of affected and
control fetuses at 96, 115, and 124/150 days gestation and from three affected
and control animal pairs at 3-5 days of age were studied by light and electron
microscopy. Morphological features evaluated included: (a) follicular structure;
(b) extent of vacuolation in specific cell types; and (c) ultrastructure of vacuoles
and follicular cells. Additionally, thyroid weights from 10 affected and 29 control
animals compiled from the B-mannosidosis research program necropsy records
were organized by age into 4 groups: 96-124/ 150 days gestation, 0-8 days, 1
month, and 4 months. Because of the small sample size in three of the four
groups, only the 0-8 day group was tested for statistical significance, using
Student’s unpaired t-test.

Two separate standardization studies were carried out. A thyroid hormone
release study sought to determine whether release was random or pulsatile and
sought to establish the validity of single, morning blood samples for thyroid
hormone assay in neonatal goats. Also, samples were collected during 14-day
neonatal periods to determine normal goat T, and T. concentrations during the
first two weeks of life and examine the possibility of abnormal thyroid hormone

concentrations in carriers of B-mannosidosis. In the thyroid hormone release

96

study, serum samples were collected every 15 minutes from four animals from
each herd at 7 days of age. The thyroid hormone release data for each animal
were analyzed by comparison of coefficients of variation to assess evidence of a
pulsatile release pattern. In the 14-day study, blood was drawn within 3 hours
after birth and then daily up to 8 days postnatally for five herd A and ten herd.
B animals; and between 9 and 14 days postnatally for five animals in each herd.
Herd and day of draw differences and interactions were assessed by a split plot
analysis of variance (19) and daily means of the two herds were compared by
Bonferroni’s t-test. To determine whether T, and T, concentrations were
influenced by sex, assay results from male and female animals between days 1
and 14 from both herds were compared by the Mann-Whitney rank-sum test.

To assess thyroid function in 8-mannosidosis, results of serum T,, free
T,, and free T, assays from six newborn affected-control pairs of animals were
compared by the Wilcoxon signed-rank test. Pairs consisted of animals 1 or 2
days old, and four of the six pairs were composed of littermates. Data were
compiled between 1983 and 1987. The upper limit of sensitivity of the T, assay

did not allow for statistical analysis of T, data.

RESULTS AND DISCUSSION
Thyroid weights
- Mean thyroid weights were greater in affected animals than in control
animals in all four age groups (Table 1). A significant difference (p<0.001)

between affected and control thyroid weights was present in the 0-8 day postnatal

97
group, the only group in which differences were compared statistically.
Morphological abnormalities in affected animal thyroids

By light microscopy, extensive cytoplasmic vacuolation was seen in most
cells of affected goat thyroid as early as 96/ 150 days gestation (Fig. 1 B).
Subjective evaluation indicated that the severity of cytoplasmic vacuolation.
increased with increasing age (Fig. 1 B,C,D). Some variation in the extent of
cytoplasmic vacuolation was present both (a) among different thyroid. sections
from any one animal and (b) among the three neonatal affected animals, but was
markedly greater in each of them than in the thyroid from the oldest (124/ 150
days gestation) affected fetus. In contrast, only occasional, isolated vacuoles were
present in control animal thyroid cells (Fig. 1, A). Affected animal thyroid
follicles were less regularly defined than control animal follicles, especially in the
oldest animals (Fig. 1 C,D). This irregularity appeared to be primarily due to the
distention of follicular cells into follicle lumina by cytoplasmic vacuolation.

By electron microscopy, cytoplasmic vacuolation was present in all cell
types in affected goat thyroid, including follicular, perifollicular, endothelial, and
perithelial cells (Fig. 2 B), and fibroblasts (not shown), compared to the normal
appearance of control animal thyroid cells (Fig. 2 A). Follicular cells contained
many more vacuoles than other cell types. Vacuole size was relatively constant
within a cell type, but varied among cell types. Endothelial cell and fibroblast
vacuoles were smaller than follicular cell vacuoles. The size of follicular cell
vacuoles in the 96/ 150 days gestation fetus was more variable than in other

affected animal cells, a variability similar to what was seen in the proximal renal

98

tubular cells of that animal (27). Vacuoles in affected animal thyroid cells were
identified ultrastructurally as lysosomal storage vacuoles (Fig. 2 C). They were
bordered by a defined limiting membrane which enclosed a floccular, amorphous
material containing occasional membrane-like structures. The occasional control
animal thyroid cytoplasmic vacuoles seen by light microscopy were typically found -
to be swollen endoplasmic reticulum cisternae by electron microscopy, and no
lysosomal storage vacuoles were encountered in normal animal thyroid cells. In
follicular cells, the appearance of both follicular cell rough endoplasmic reticulum
and luminal aspects of plasma membrane surfaces were morphologically similar
in affected and control thyroids at all ages. Colloid was ultrastructurally similar
in affected and control animals.

Perifollicular cells, identified ultrastructurally, were only infrequently
found in affected animal thyroids. The lysosomal storage vacuoles in those
perifollicular cells clearly identified by secretory granules differed from follicular
cell vacuoles in that they were both generally smaller in size and less numerous.
Occasional affected goat thyroid cells with a lysosomal storage vacuole
appearance similar to perifollicular cells but depleted of secretory granules were
noted (large arrow, Fig. 2 B). No comparable secretory granule-depleted
perifollicular cells were seen in control thyroid.

Thyroid hormone release in neonatal goats

The .4-hour pattern of thyroid hormone release in 7-day-old goats (Fig. 3)

was relatively constant for all four animals from both herds. The average

coefficient of variation among the eight animals was 0.07 (range 0.08 to 0.05) for

99

T, and 0.08 (range 0.15 to 0.06) for T,. Thus, there was no evidence of a pulsatile
release pattern. In the 14-day evaluation of newborn animal thyroid hormone
concentrations, serum T, concentration decreased significantly (p<0.05) during
the first week of life and then leveled off in both herds (Fig. 4 A). Also, T,
concentration in herds A and B differed significantly (p<0.05) between 2 and
6 days of age. T, concentration in both herds were relatively stable during the
first two weeks of life, and did not vary significantly between herds (Fig. 4 B).
Neither T, nor T, varied significantly with sex.
Affected and control animal thyroid hormone concentrations

Comparison of T,, FL, and PT, concentrations in serum drawn from six
age-matched affected-control animal pairs (Fig. 4) found affected animal values
to be significantly lower (p < 0.05) than control values. For the six pairs, there was
an average reduction in affected animal T, concentration to 66% of control
concentration. T, concentration exceeded} the highest standard in four pairs and
could not be analyzed statistically.
Discussion

This study represents, to our knowledge, the most complete description ‘
of developmental changes and the first report of abnormal function in an
endocrine organ in a lysosomal storage disease. Further, this is the first report
suggesting that the pathogenesis of central nervous system (CNS) lesions in a
storage disease could involve endocrine perturbations. Many storage diseases
have associated clinical and morphological CNS abnormalities, but the impact of

the storage disease on the CNS is poorly understood and has often been

100
attributed to direct effects of accumulated substrate (42,45).

Some of the increased thyroid weight in affected animals may be
explainable by the extreme degree of lysosomal storage in the organ. In a
previous study, thyroid from an affected goat was found to contain 9.9 mg of
the predominant storageoligosaccharides per gram wet weight of thyroid (18),.
constituting only about 1% of the weight of tissue. Although this value does not
take into consideration the weight of the water likely to be found in lysosomal
storage vacuoles along with the oligosaccharides, storage of oligosaccharides
probably does not by itself account for all of the increased mass of affected
animal thyroids. The possibility of increased thyrotropin concentration, which
may lead to thyroid hyperplasia, has not yet been assessed in affected animals
because of lack of a TSH assay that detects the goat hormone.

Little information is available in the literature concerning thyroid function
in neonatal goats, although a number of studies have reported thyroid hormone
concentrations in goats 3 weeks old or older (28,39,44). The timing of blood
draws with respect to hormone secretion patterns 'can be important for accurate
assessment of thyroid hormone concentrations. This is of particular importance
in neonatal animals, since thyroid hormone concentrations are generally high at
birth and drop substantially during the first week of life (16). The present study
demonstrated that T, and T, concentrations in neonatal animals are relatively
constant within a 4-hour period and no evidence of pulsatile release was present
in the thyroid hormone release study (Fig. 3). Thus, single morning samples,

during at least the first week of life, should be representative of baseline thyroid

101

hormone concentrations and not an artifact of pulsatile release. The significant
difference in T, concentration between the herds during the first week of life in
the 14-day study was an unexpected finding (Fig. 4A). Factors that may help to
explain the differences include (a) induction of parturition in herd B but not in
herd A, with herd B animals 2 to 3 days younger in gestational age at delivery.
than herd A animals; (b) environmental differences, such as differences in feed
eaten by the lactating does; and (c) B-mannosidosis carrier status or other herd
genetic factors. Nonetheless, the results indicate that no evidence of carrier goat
thyroid hypofunction is present.

Results from thyroid hormone assays of affected-control pairs compiled
between 1983 and 1987 indicate that thyroid hormone concentrations are
significantly reduced in affected animals. Since lysosomal storage vacuoles occupy
a major proportion of neonatal thyroid follicular cells, it is somewhat surprising
that a T, concentration of even 65% of normal is achieved. The absence of
validity testing of the T,, FT, and FT, assays with normal goat serum is a
weakness in this study. However, extensive comparisons of canine T,
concentration using both the Becton Dickinson T, kit, used in the affected-control
pair study (no longer available as originally formulated), and the Ciba Corning
T, kit, currently in use, found substantially similar readings (unpublished data).
Additionally, a recent 1-day-old affected animal had a T, level of 89.5 nmol/l,
similar to the values for the two 1-day-old affected animals in the pair study.
Thus, the data suggest that affected animals have deficient thyroid hormone

concentrations, and indicate that further investigation of thyroid function is

102
warranted.

Central nervous system (CNS) hypomyelination, a major pathological
feature of caprine B-mannosidosis, is characterized by regional variation in extent
of hypomyelination (31). Results of several studies have suggested that
developmental defects may lead to the reduced number of oligodendrocytes.
present in myelin deficient CNS regions of affected animals (25,30-32). Thyroid
hormones play an important role during development of the central nervous
system (1,20,21) and considerable evidence indicates that thyroid hormones
have a direct impact on myelination (21,46,48). Hypothyroidism has been
associated with deficient myelination (46) and impaired oligodendrocyte
differentiation and function (2,8,9,11,12,33,36,41). In addition, in border disease,
a disease of sheep caused by in mono infection of fetuses by the border disease
virus (BDV) (4-7,37), a reduced thyroid hormone level, possibly secondary to
follicular cell invasion by BDV, has been postulated to be the causative factor
in the disease’s severe CNS myelin deficiency (3). Average T, and T,
concentrations in border disease sheep were 60% and 74% of control values,
respectively, similar to the extent of reduction in B-mannosidosis. Thus, thyroid
hormone deficiency in goats affected with B-mannosidosis may be a major factor
in the pathogenesis of hypomyelination. Whether the reduction in thyroid
hormone concentrations seen in newborn affected goats is indicative of a
comparable reduction in gtgo, and whether a reduction in T, concentration. to
65% of control is enough to impair myelination to the extent seen in the cerebral

hemispheres of affected goats remains to be determined. If vacuolation is either

103

by itself the major factor in decreased thyroid hormone concentrations or is
symptomatic of metabolic perturbations that might disrupt thyroid function, the
increased vacuolation during development may suggest that a progressive
decrease in hormone release takes place as development proceeds. Decreasing
concentrations of thyroid hormones during development could explain the.
generally increased severity of myelin deficits in regions that myelinate late in
CNS development (31).

Thyroglobulin is a glycoprotein and contains Man81-4GlcNAc bonds in its
N -linked oligosaccharides (43,49). The direct impact of B-mannosidase deficiency
on thyroglobulin metabolism would occur during lysosomal catabolism of
thyroglobulin, and a degradation impediment or defect is the most likely
alteration that would lead to decreased thyroid hormone secretion in 8-
mannosidosis. Absence of affected goat lysosomal B-mannosidase activity and
accumulation of oligosaccharides could have an adverse effect on thyroglobulin
degradation, and then on thyroid hormone release, in several ways, including
through (a) decreased availability of primary lysosomes due‘ to impaired recycling
of secondary lysosomes (13,40), and (b) interference in cell function by storage
vacuole accumulation. Relevant to both possibilities, a recent study identified a
possible post-endocytosis regulation step in thyroglobulin catabolism, involving
a lag period between fusion of endosomes with primary lysosomes to form
secondary lysosomes (40). Alterations in the availability of primary lysosomes,
due to alterations in synthesis or in accessibility, might adversely affect post-

endocytosis regulation. Thyroid metabolic alterations other than disruption of

104

catabolism-such as a thyroglobulin synthesis defect, an organification defect, a
hormone receptor abnormality, or a thyrotropin deficiency--are much less likely
to be involved in the thyroid hormone deficits in B-mannosidosis. It is possible,
however, that metabolic perturbations secondary to abnormal glycoprotein
metabolism could adversely affect thyroglobulin metabolism. The thyroid in 8-
mannosidosis may provide a good system in which to study the role of lysosomes
in thyroglobulin metabolism.

Little information is available about endocrine morphology and function
in storage diseases. Some thyroid pathological changes have been reported in
Cl-mannosidosis, the lysosomal storage disease most closely related to B-
mannosidosis. Thyroid epithelial cell vacuolation was not found neonatally in
neonatal bovine cut-mannosidosis (24), but vacuolation has been reported in the
thyroid of cattle which ingested the a-mannosidase inhibitor swainsonine (23).
Additionally, in feline a-mannosidosis, the thyroid was found to have the greatest
storage of oligosaccharides, per unit wet weight, of any tissue analyzed (47).
Thyroid hormone serum concentrations have not been reported in o -
mannosidosis or in swainsonine poisoning.

In this study, the extensive and developmentally progressive morphological
abnormalities present in the thyroid of goats affected with B-mannosidosis are
documented. Additionally, evidence suggesting deficient thyroid function in
affected goats is presented. This functional abnormality may be due, at least in
part, to the extensive thyroid follicular cell morphological abnormalities or to

metabolic perturbations secondary to deficient B-mannosidase activity. Thyroid

105

hormone deficiency in sheep has been proposed to cause clinical features such
as doming of the skull, limb abnormalities, and hypomyelination, each prominent
abnormalities in caprine B-mannosidosis. Thus, a role for reduced thyroid
hormone concentrations in the myelin deficits and other clinical and pathological
abnormalities seen in goats affected with B-mannosidosis is possible and needs.

further investigation.

106
METHODS

Animal acquisition

Animals were obtained from two goat herds: herd A, consisting of normal
goats, and herd B, consisting of carrier goats. In herd A, pregnancies were
established by natural breeding of cycling does, parturition was natural, and.
gestation time varied from 146 to 150 days. Pregnancies in herd B were
established in a planned parturition program with natural breeding. Doe estrus
cycles were synchronized using sponges impregnated with 45 mg fluorogestone
acetate (Intervet, Boxmeer, Netherlands) and ovulation was induced by injection
of 600 IU PMSG (Intervet) (15). Fetal animals, all from herd B, were delivered
by cesarean section. Parturition in herd B was induced by injection of does with
0.5 cc Estrumate (Butler, Brighton, MI), a synthetic prostaglandin Fn analog, at
day 143, and delivery was at 144-145 days gestation.
Tissue preparation

Thyroid samples, collected after T-61 (American Hoechst, Somerville, NJ)
euthanasia, were fixed in 4% glutaraldehyde, post-fixed in 1% osmium tetroxide,
processed routinely, and embedded in an Epon-Araldite mixture. For light
microscopy, semithin sections (1 um) were cut from between four and eight
blocks per animal and stained with toluidine blue. For electron microscopy,
ultrathin sections were cut from at least two blocks per animal and stained with

uranyl acetate and lead citrate.

107

Thyroid hormone assays
For 4-hour and 14-day studies, serum T, and T, concentrations were
estimated by radioimmunoassay (RIA). For T, assay, a Ciba Corning Magic kit
was used. Twenty five ul of standard or unknown sample and 100 111 of kit
tracer/buffer solution were pipetted into polystyrene tubes and 500 111 of.
antibody—buffer solution was added to all tubes except the total count tubes. The
contents of the tubes were mixed by vortexing and the tubes were incubated at
room temperature for 1 h. The tubes were placed in magnetized racks for 5
minutes to separate the antibody-bound fraction from the free fraction. The
liquid content of all tubes except the total count tubes were poured off and the
tubes were allowed to drain on absorbent paper for 2 minutes. Antibody-bound
“’1 was quantified with a gamma emission spectrometer. Assay sensitivity was
estimated at 13 nmol/L, the point of 90% of total binding on the standard curve.
Assay specificity was determined with a serial dilution method. Dilution of
caprine sera with 0 standard paralleled standard curves, but dilution with distilled
water or gelatin-phosphate buffer underestimated expected T, indicating a matrix
effect. When 10, 25, 50 or 100 ng/ml of T, were added to caprine serum to
determine assay accuracy, 100, 101, 100, and 97% of exogenous T, was measured,
respectively. Intraassay coefficients of variation (CV) for two caprine control
sera were 1.1% (mean = 185 nmol/L, number of assays = 3) and 3.3% (mean
= 167 nmol/L, number of assays = 3). Interassay CV’s for the same sera were
1.7% and 3.8% respectively (number of assays = 3). Procedures for the TT,

radioimmunoassay have been described previously for bovine sera (38). The

108

antiserum was used at a final dilution of 1:6000 in assay buffer. The assay
sensitivity was estimated at 0.02 nmol/L, the concentration calculated at 90% of
total binding. Dilution of caprine sera paralleled standard curves. When 0.25, 0.5

or 1.0 ng of T, was added to caprine serum to determine assay accuracy, 102%,

101%, and 101% of exogenous hormone was measured in the assay, respectively. .
Dilution of caprine serum with 0 standard paralleled standard curves, but dilution
with distilled water or gelatin-phosphate buffer indicated that parallelism was lost
at the 1:8 dilution. Intraassay CV’s for two caprine sera were 21.5% (mean =

4.15 nmol/L, number of assays = 3) and 4.0% (mean = 4.3 nmol/L, number of
assays = 3). Interassay CV’s for the same serum pools over 3 assays were 6.2%
and 7.9%, respectively.

Affected-control pair assays of T,, free T,, and free T, concentrations, all
run between 1983 and 1987, were made using Becton Dickinson assay kits.
Validity testing with goat serum was not performed with these assays, however,
extensive assessment of canine T, concentration using both the Becton Dickinson
T, kit (no longer available as originally formulated) and the Ciba Corning T, kit
found substantially similar readings (unpublished data). The T, assay was the

same as described above.

109

Acknowledgements:

I wish to acknowledge the guidance of Drs. Kathryn Lovell and Margaret Jones
with this project and their assistance with revisions of the manuscript. Dr.
Raymond Nachreiner helped plan and implement the thyroid function study, Dr. .
Kent Refsal did the statistical analysis of the 4-hour and 14-day study data, and
both assisted with revision of the manuscript. Cesarean sections were performed
by Dr. N. Kent Ames and anesthesia was administered by Dr. Cheryl Blaze, both
from the Michigan State University Department of Large Animal Clinical
Sciences. The excellent technical assistance of Ralph Common, Nancy Truscott,
Jayne Kelley, Robert Kranich, Joanne Klug, Carol Ayala, and Jane Benke is

much appreciated.

This work was Supported by National Institutes of Health Grants NS-20254
(KLL), NS-16886 (MZJ), and RR-05655.

110

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TABLE 1. MEAN THY ROID WEIGHTS AT VARIOUS STAGES OF DEVELOPMENT

 

Control Affected

 

 

Age n weight (g) n weight (g)

 

96-120/150 5 0.36 (0240.42)b 2 0.82 (0.73-0.90)
Days Gest.‘

0-8 Days 15 0.68 (0.51-1.00) 5 1.17'3 (0.93-1.45)
1Month 4 0.32 (0590.95) 2 2.12 (2.03-2.20)
4 Months 5 2.29 (1.42-3.00) 1 3.00 -

 

‘ days gestation
" range
‘ p < 0.001

118

TABLE 2. THYROID HORMONE ASSAY SUMMARIES FROM SIX AFFECTED
AND CONTROL ANIMAL AGE-MATCHED PAIRS

 

 

Age s-Man. T, T, FT, FT,
(days) Status‘ (nmol/l) (nmol/l) (pg/ml) (pg/ml).

1 C 133.3 >6” 22.8 -‘
A 92.7 > 6 12.7 -

1 C 1 1 7.5 > 6 14.5 7.9
A 100.6 > 6 10.3 5.9

2 C 91.9 - - 6.9
A 51 .9 - 4.5 5.3

2 C 96.1 6.86 18.7 16.1
A 63.5 5.94 12.4 10.6

2 C 95.6 6.48 13.1 11.1
A 55.3 2.8 3.9 3.0

2 C 89.3 5.2 10.4 8.6
A 49.8 3.3 4.1 3.5

 

: 3-mannosidosis status: C, control; A. affected
value exceeded highest standard value
.° indicates test not done or insufficient sample size to run test

119

FIG. 1. Light micrographs of toluidine blue stained lum Epon sections from
control and affected animals at 96/ 150 days gestation (A,B), and affected animals
at 124/ 150 days gestation (C), and 3 days postnatal (D). Extensive vacuolation
was present in affected goat thyroid cells as early as 96/ 150 days gestation (B),

and became more pronounced with advancing age (CD) 275 X.

120

 

121

FIG. 2. Electron micrographs of thyroid follicular cells from 3-day-old control
(A) and affected (B,C) goats. Control'animal thyroid follicular and perifollicular
cells (A, small and large arrows, respectively) contained only occasional
cytoplasmic vacuoles. A large number of cytoplasmic vacuoles were present in
all affected animal thyroid cell types (B) including follicular cells (small arrow),
endothelial cells (arrowhead), perithelial cells (double arrow), and perifollicular
cells (large arrow), shown degranulated here. Vacuole size and number varied
among cell types. Higher magnification demonstrated that cytoplasmic vacuoles
in affected animal thyroid cells were consistent with lysosomal storage vacuole
structure (C). Their limiting membrane (arrows) was distinct from adjacent rough

endOplasmic reticulum. A,B 4,940 X; C 27,000 X.

122

 

123

FIG. 3. T, and T, release pattern assessed in serum samples drawn at 15 minute
intervals over 4-hours, between 8:00 am and 12:00 noon, from a representative.

herd A (normal animals) and herd B (carriers of B-mannosidosis) animal.

FIG. 4. T, (A) and T, (B) mean values from blood draws at birth (day 0) and
between days 1 and 14 postnatal from herds A (n = 5) and B (n = 10, days 0-
8; n = 5, days 9-14). Error bars show standard deviation. " indicates statistically

significant difference (p<0.05) between herds A and B at indicated age

FIG. 3
100. I----I Herd A
9° " o——+ Herd e T 4
S
O
E
5
£2
0
>
O
..I
F!
'0
r:
I!
1.5.”
O 1 1 1 1 1 J L 1 1 1 1 1 1 1W1 1 L
8:00 9:00 10:00 11:00 12200
Time of Draw (am)
FIG. 4
300 ,
c A l---l Herd A
3 250.- -
g H Herd B
5
.59.
d)
>
0
._I
1.:
,C
«I
d)
2
O 1 1 1 1 1 1 1 L 1 n _1 1 1__
1 2 3 4 5 6 7 8 9 10 12 14
Age (days)
‘i B
g : I---I Herd A
O
E ' 0—0 Herd 8
5
.‘2
d)
>
d.)
_I
I"?
C.
(D
0)
>2 .
l'
O 1 1 1 1 1 1 1 1 1 1 _1 L 1

 

124

 

 

 

 

 

 

 

 

0-12 3 45678 9101214
Ageidays)