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Ultrastructural and metabolic changes associated with reproductive tract atrophy and adiposity in diabetic female mice.

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THE ANATOMICAL RECORD 216:359-366 (1986)
Ultrastructural and Metabolic Changes Associated
With Reproductive Tract Atrophy and Adiposity in
Diabetic Female Mice
DAVID R. GARRIS, R. LEE WEST, AND PHILLIP H. PEKALA
East Carolina University, School of Medicine, Greenville, NC 27834
ABSTRACT
The effect of progressive, diabetes-associated adiposity on reproductive tract structure and function was examined in 4- to 16-week-old C57BL/KsJ,
control (+/?) and diabetic (db/db) mice. Uterine and ovarian tissues were analyzed
by transmission electron microscopy for ultrastructural changes associated with
increased intracellular lipid accumulation. In addition, the same tissues were analyzed for changes in activity of tissue lipoprotein lipase, an enzyme that hydrolyzes
lipoprotein-associatedtriacylglycerols and supports the cellular uptake and storage
of free fatty acids. Between 8 and 16 weeks of age, intracellular lipid deposits
increased dramatically in the ovarian granulosa, thecal and stromal cell populations, as well as in the uterine epithelium, of diabetic mice compared to controls. By
16 weeks of age, the lipid deposits essentially occupied the entire cytoplasmic area
of both the ovarian and uterine cell types in diabetics. The basal lamina underlying
the uterine epithelium was expanded in the diabetics relative to controls, and the
hyperglycemic condition induced an observable increase in endometrial intercellular
space that was occupied by a hyaline type of ground substance of unknown composition and origin. In association with these structural changes, both ovarian and
uterine lipase activities were greatly increased in the dbldb mice compared with
controls. These data suggest that the structural adiposity and functional decline in
reproductive tract condition of the dbldb mutants are related to the enhanced
cellular lipid deposition observed in this species. These changes in structural and
metabolic parameters are related to the reproductive incompetence characteristic of
this murine model.
A decline in female reproductive performance is a
recognized consequence of the diabetes syndrome in
many mammalian species, including humans (Shipley
and Danley, 1947; Lawrence and Cantopoulos, 1960;
Chieri et al., 1969; Williams and Porte, 1974). In association with both type I (i.e., insulin-dependent) and type
11 (i.e., non-insulin-dependent) diabetic states, changes
in the structure, metabolism, responsiveness, and function of the female reproductive tract tissues have been
described (Garris et al., 1982, 1984a, 1985a; Garris and
Smith, 1983; Johnson and Sidman, 1979; Vomachka and
Johnson, 1982). Depending on the severity of the diabetic condition, anovulation (Shipley and Danley, 1947;
Kirchick et al., 1978), altered cyclic patterns (Chieri et
al., 1969),depressed follicular recruitment (Garris et al.,
1985b), insensitivity to exogenous gonadotrophin therapy (Kirchick et al., 1978; Vomachka and Johnson, 1982),
depressed ovarian steroidogenesis (Vomachka and Johnson, 1982; Garris et al., 1982, 198513))incomplete luteal
development and function (Garris et al., 1984a; Garris,
1984), as well as ovarian atrophy (Davis et al., 1947;
Lawrence and Cantopoulos, 1960; Garris, 1984)occur in
association with the hyperglycemic state. In addition,
changes in uterine sensitivity to gonadal steroids (Garris, 1985), structural organization of the epithelial and
stromal layers (Garris, 1985; Garris et al., 1984b; Garris
0 1986 ALAN R. LISS, INC.
and Smith, 1983),tissue carbohydrate metabolism (Garris et al., 1985a; Swigart et al., 196l), and responsivity
to stimulation (Kirkland et al., 1981) characterize the
condition of the reproductive tract in diabetics. These
changes in utero-ovarian parameters have been associated with the recognized decline in fetal-placental
maintenance during pregnancy in diabetic females (Hall
and Tillman, 1951; Sinden and Longwell, 1949). In addition, these structural and functional changes in the
reproductive tract become more pronounced with the
increased duration of the diabetic condition (Garris,
1984; Garris et al., 1984b).
The diabetes (db/db) mutation on chromosome 4 in the
C57BLKsJ mouse induces a hyperglycemic, hyperinsulinemic and obese murine model that experiences reproductive tract atrophy (Coleman, 1978; Garris et al.,
1985~).
Recent studies indicate that the diabetes-induced
changes in both utero-ovarian structure and function
(Garris, 1985; Garris et al., 1985~)result in cellular
involution and tissue atrophy associated with a pronounced intracellular adiposity (Garris et al., 198%).
Between 8 and 16 weeks of age, the ovarian thecal,
stromal, and granulosa cells, as well as uterine epithelium of diabetic mice exhibited a high concentration,
Received January 8,1986; accepted April 21,1986.
360
D.R. GARRIS, R.L. WEST. AND P.H. PEKALA
compared to controls, of intracellular lipid that was demonstrated by differential staining (Garris, 1985; Garris
et aI., 1985~).
The lipid accumulation was temporally
associated with pronounced tissue atrophy and dysfunction and was suspected to be associated with the infertility problems common to this diabetes mutant. Similar
changes in reproductive tract function observed in genetically obese rats have been linked to abnormal cellular lipid synthesis and activity of lipoprotein lipase (Gray
and Greenwood, 1983, 1984), an enzyme that metabolizes triacylglycerols and permits the uptake and storage of free fatty acids in nonhepatic tissues. To date, an
ultrastructural and metabolic examination of the cellular changes that occur in association with the progressive adiposity and atrophy of the reproductive tract of
the db/db mutants has not been described. The present
studies were undertaken to correlate the ultrastructural
changes that occur in uterine and ovarian cell types
with the related alterations in lipoprotein lipase activity
and lipid accumulation in these peripheral tissues.
MATERIALS AND METHODS
Animals
Adult, female C57BL/KsJ mice were obtained from
the Jackson Laboratory (Bar Harbor, ME). Both control
(+/?) and diabetic (db/db) genotypes were match-paired
at 4,8, and 16 weeks of age. All mice were housed under
controlled environmental conditions (23-25 "C) with an
established photoperiod of 12 hr light per day (lights on:
0600 hr). Body weights and blood glucose concentrations
(Technicon Autoanalyzer method) were determined at
the time of sacrifice for all mice. All db/db mice that
were obese andor hyperglycemic relative to age-matched
controls by 4 weeks of age were considered diabetic
(Table 1).
Tissue Collection and Preparation
The uteri and ovaries were collected from 8- and 16week-old control and diabetic mice for analysis by transmission electron microscopy (TEM). As previously described (Garris et al., 1982, 1984b), mice were
anesthetized with sodium pentobarbitol and perfused
with 50 ml of physiological saline (by intracardiac puncture), which was followed by 100 ml of Karnovsky's
fixative solution. The tissues were cleaned, blotted,
blocked, and embedded in plastic by conventional techniques. All tissues were subsequently sectioned and
stained with osmium tetroxide prior to examination by
TEM. All uterine specimens examined were from the
midcornua zone. Ovarian cell changes were analyzed
from specimens containing secondary follicles that exhibited the structural characteristics of viable follicles,
as previously described (Garris et al., 1985~).
Tissue Lipoprotein Lipase Assay
Groups of mice corresponding to those used for TEM
analysis were sacrificed by cervical dislocation and the
ovaries, uterus, pancreas, and liver were removed and
weighed. Each tissue was immediately rinsed in sterile
Dulbecco's modified Eagles (DME) medium containing
2% bovine serum albumin, then blotted on sterile filter
paper. The tissues were subsequently minced and placed
into sterile polypropylene culture tubes containing 1mi
of DME medium supplemented with 2% bovine serum
albumin and 10 U of heparin. Tubes were then capped
and incubated a t 37°C in a 90%02110%C02 atmosphere
for 2 hr with gentle shaking. At the end of the incubation period, each tube was centrifuged (3,OOOg/ 30 min)
and the supernatant was collected and used as the source
of the tissue lipoprotein lipase in the assays.
Assays for lipoprotein lipase were performed in triplicate pools within 30 min after the preparation of the
tissue lipase samples according to the method of NilssonEhle and Schotz (1976). In brief, 75 p1 of the isolated
enzyme preparation was mixed with 25 p1 of a substrate
that consisted of 22.6 mM (3H)-triolein (1.4 pCdpmol),
lecithin (2.5 mg/ml), bovine serum albumin (40mg/ml),
33% (vol/vol)human serum and 33%(vol/vol)glycerol in
0.27 M Tris-HC1(pH: 8.1) buffer and incubated at 37°C
for 90 min. The reactions were subsequently terminated
and the free fatty acids (FFA) were separated from the
incubation mixture by liquid-liquid partition extraction
as follows. To each tube, 3.25 ml of a methanol : chloroform : heptane (1.41 : 1.25 : 1vollvol) mixture was added,
followed by 1.05 ml of a 0.1 M potassium carbonateborate buffer (pH 10.5). The combined solution was vigorously mixed and centrifuged at 3,OOOg for 15 min at
23°C. An aliquot (300 pl) was obtained from the methanol-water upper phase, which contained the free fatty
acids. Radioactivity present in the free fatty acid fraction was determined by liquid scintillation analysis. One
milliunit of enzyme activity was defined as the release
of 1 nmol of fatty acid per 1 min, and all values were
then expressed as nanomoles FFN1 m i d l gm tissue.
The activity in all cases was inhibited by either the
addition of 1 M NaCl or by the omission of serum from
the assay. All values were expressed as group means
(+SEMI with intergroup differences (P < 0.05) determined by the Student t test for each tissue group
analyzed.
RESULTS
and blood glucose concentrations in C57BLMsJ mice
The uterine epithelium of 8- to 16-week-old control
Body weight
Blood glucose mice appeared as a pseudostratified-to-simplecolumnar
Age
cell type with prominent, indented nuclei forming a
(mgldl)
(weeks)
Group
N
(gm)
well-organized
nuclear zone (Fig. 1A).The apical borders
4
(+I?)
5
17.2 + 0.1
159.6 + 1.3
(db/db)
5
22.8 + 0.2*
148.3 + 3.1
6
18.7 + 0.4
90.6 + 4.7
8
(+I?)
4
38.0 + 0.4*
406.8 + 24.1*
(dbldb)
4
24.0 + 0.7
90.3 + 9.6
Fig. 1. Representative photomicrographs ( X 2,100) of the uterine
16
(+I?)
502.2 + 13.2* epithelium in 8- to 16-week controls (A) and in 8-week-old(B) and 1650.8 + 0.7*
(db/db)
5
TABLE 1. Age- and diabetes-related changes in body weight
week-old (C) diabetic mice. Lipid deposits (L)were visible in all groups,
All values are represented 88 group means (+SEMI for control (+/?) the most abundant concentrations being apparent in the diabetic mice.
and diabetic (db/db) C57BL/KsJ mice. Significant differences between The basal lamina (BL) in the diabetic mice was thickened and the
genotypes of each age group are denoted by an asterisk 8 < 0.05).
basal pseudopodia emanating from the inferior epithelial surface were
obvious.
DIABETES AND REPRODUCTION
361
Fig. 2. Photomicrographs of the basal pole of control (A) ( X 7,000) diabetic 03) ( X 10,500) uterine
epithelium demonstrating the high concentrations of lipid (L)deposited in this zone in 8-week-olddiabetic
mice. The basal lamina (BL) underlying the epithelial layer surrounded the basal pole pseudopodia, which
were common in the cells of diabetic mice. Periluminal stromal cells (S) remained in close apposition to
the basal lamina. The nuclei (N) of the epithelial cells were prominent in all control and diabetic
specimens.
363
DIABETES AND REPRODUCTION
presented a thick, microvillous surface, whereas the
basal membrane surface appeared smooth and adherent
to the underlying basal lamina (Fig. 2A). A small
amount of intracellular lipid was noted in the basal zone
of the epithelial cells from typical 16-week-old control
mice (Fig. 1A). In contrast, the uterine epithelium of
both 8-week-old (Fig. 1B) and 16-week-old(Fig. 1C) diabetic mice exhibited very prominent, and expanded, lipid
accumulations in all of the samples examined. In addition, lipid droplets were observed scattered throughout
the cytoplasm of the epithelial cells from diabetic mice,
the increased cellular lipid content being most prominent in the 16-week-oldmice. The basal lamina supporting the epithelial layer in diabetics was expanded (Fig.
2B) relative to controls. Basal membrane evaginations
from the epithelial cells of the diabetic mice served to
anchor the cellular layer to the endometrial basal lamina (Fig. 2B).
In control mice, the ovarian granulosa layers were
composed of ovoid cells with large, prominent nuclei and
a mitochondria-rich cytoplasm (Fig. 3A). In contrast, the
granulosa cells in the cumulus zone of 16-week-olddiabetic mice (Fig. 3B) contained an enormous amount of
lipid, which occupied nearly the entire intracellular area
of most cells. In cells where nuclei could be identified
(Fig. 3B), the nuclear membranes appeared indented
and shrunken relative to controls. In most specimens,
little structural detail of the intracellular organization
of the granulosa cells could be determined owing to the
heavy lipid deposits present in the cells of diabetic mice.
In contrast t o the ordered layers of thecal and stromal
cells surrounding the granulosa layers of control ovaries
(Fig. 4A), the stromal cells of diabetic mice appeared t o
be undergoing degenerative changes between 8 and 16
weeks of age. The nuclei of the stromal cells were less
prominent and smaller in the diabetics than in controls
(Fig. 4B). In addition, there was an obvious lack of mitotic activity in the cells examined and a dispersed intranuclear chromatin pattern. By 16 weeks of age (Fig.
4C), the orderly array of periluminal stromal cells observed in control specimens was absent in the diabetic
tissue samples. The cytoplasmic membranes of the dbl
db stromal cells were indented and degenerative in appearance compared t o the smooth-surfaced features of
controls (Fig. 4A). There was an apparent increase in
the intercellular space between stromal cells in the diabetic endometrium that was occupied by a hyaline type
of ground substance of unknown composition or origin
Fig. 4C). No evidence of functional, intracellular organelles was observed in the periluminal stromal cells of
16-week-olddiabetic mice.
Between 8 and 16 weeks of age, a dramatic increase
in lipoprotein lipase activity was observed in the uteri
and ovaries of diabetic mice relative to controls (Table
2). By 8 weeks of age, the lipase activity was approximately 1.5-5 times greater in the tissues collected from
diabetic mice than in those from controls. By 16 weeks
of age, these changes became even more pronounced as
both the uteri and ovaries of the diabetics became infiltrated with lipid deposits. The continued increase in
lipase activity in the ovarian and uterine tissues of the
diabetic mice correlated temporally with the progressive
increase in intracellular lipid deposits observed in the
corresponding tissues examined by TEM.
DISCUSSION
The results of the present studies indicate that the
diabetes-related decline in reproductive tract function in
dbldb mice is associated with an increased intracellular
lipid deposition in uterine epithelial cells and in ovarian
granulosa, thecal, and stromal cells. The observed elevation in lipoid deposition was temporally related to the
dramatic increase in tissue lipoprotein lipase activity in
the diabetic mice compared with controls. These changes
in cellular structure and metabolism were progressive
with respect to the age of the dbldb mice, the 16-weekold groups exhibiting exaggerated cellular atrophy compared to the 8-week-oldmice. By 16 weeks of age, all of
the ovarian cell types in the dbldb mice were completely
infiltrated with lipid, a condition that we term the “fatty
follicle syndrome” in this murine model. The condition
apparently underlies the functional decline in ovarian
activity that is characterized by an increased follicular
atrophy, depressed luteal function, declining steroidogenic activity, and general structural degeneration of
all cell types in this mutant (Garris et al., 1985~).
Similar structural and metabolic changes in reproductive
tract activity are common in other obese and diabetic
rodent models (Saiduddin et al., 1973; Swerdloff et al.,
1976; Chieri et al., 1969; Garris et al., 1982, 1984a;
Garris, 19841, suggesting that the functional impairments in utero-ovarian activity in these species may
have a common cellular deficit or incompetence for adjusting to the changes in systemic lipid and carbohydrate metabolism that characterize these models.
The changes noted in tissue lipase activity in the present study support the reported presence of lipoprotein
lipase activity in the uterus of obese Zucker rats (Gray
and Greenwood, 1984) and the relationship between altered enzyme activity and reproductive problems in
these animals. In both species, tissue lipase activities
were altered in the reproductive tract tissues of these
genetic mutants compared to controls. Estrogens have
been demonstrated to modulate cell lipase activity (Gray
and Greenwood, 1984)and in consequence possibly influence the rate of triglyceride uptake by gonadal steroidsensitive cell types in the ovary and uterus. The depressed circulating estradiol levels in diabetic mice
(Garris et al., 1985~)
may account for the elevated lipase
activity noted in the present studies, since estradiol
depresses lipase activity in the uterus of obese subjects
(Gray and Greenwood, 1984). Recent evidence suggests
that estrogens and related metabolites are also capable
of modulating circulating glucose levels and related tissue parameters in the dbldb mouse and in the obese
Zucker rat (Coleman et al., 1982; Gansler et al., 1985).
Thus, the corrective effects induced by estradiol with
respect to cellular metabolism may occur through the
modulation of lipid catabolism, as well as carbohydrate
utilization.
Alterations in pancreatic (Coleman, 19781, renal (Like
et al., 1972), CNS (Garris et al., 1985a,b), peripheral
nerve (Hanker et al., 1980),and hepatic (Coleman, 1982)
structure and function are recognized to occur in association with the diabetes syndrome in this murine model.
The changes are influenced by the duration of the hyperglycemic-hyperinsulinemic condition that characterizes
the expression of the genetic mutation in this murine
364
D.R. GARRIS, R.L. WEST, AND P.H. PEKALA
Fig. 3.Photomicrographs ( x 3,812) of the granulosa cell layers of antral follicles from control (A) and
diabetic (B) mice between 8 and 16 weeks of age. All cells from control mice exhibited distinct nuclei (N)
and a mitochondria-rich cytoplasm. In contrast, the lipid deposits (L) present in the granulosa cells of
diabetic mice essentially obliterated the cytoplasmic space. The nuclei present in the granulosa cells of
diabetic mice appeared degenerative.
365
DIABETES AND REPRODUCTION
model. Changes in the reproductive-neuroendocrineaxis,
which modulates ovarian activity, have also been reported for this species (Johnson and Sidman, 1979; Garris et al., 1985b). Hence the abnormal metabolic and
endocrine regulation of cellular function in the dbldb
mice probably contribute to the reproductive tract involution observed in the present studies. However, preliminary studies (Garris, unpublished observations) suggest
that, even after prolonged exposure to the adverse metabolic conditions that characterize this mutant, uterine
and ovarian tissues will respond to endocrine replacement therapies, as indicated by normalized structural
and metabolic parameters. These data suggest that the
therapeutic normalization of the peripheral metabolic
disturbances in this murine mutant may also correct or
prevent the diabetes-associated depression of female reproductive tract function.
The metabolic and structural changes that occur in
uterine or ovarian cells of untreated human diabetics
are not well understood. Since the availability of insulin
for the treatment of diabetes in the mid-l920s, most
humans are treated for the overt symptoms of the disease as soon as it is clinically detected. However, Krause
(1936)reported that both cellular atrophy and adiposity
characterized the ovarian cellular components collected
from a small number of untreated, diabetic females. The
structural changes described by Krause (1936)resemble
those common to the db/db murine mutant, suggesting
that this model may be useful in the testing of therapeutic approaches designed to correct the reproductive dysfunction that characterizes both rodent and human
diabetics.
In summary, the results of the present study indicate
that the cellular accumulation of intracellular lipid by
TABLE 2. Lipoprotein lipase activity in C57BLMs.J mice:
Effects of age and diabetes (db/db) mutation
Age
(weeks)
8
Grour,
(+I?)
(db/db)
(+/?)
(db/db)
16
(+/?)
(db/db)
(+I?)
(db/db)
N
Tissue
Lipase activity
(nmoles FFMmidmn)
5
5
5
5
4
5
4
5
Uterus
Uterus
Ovary
Ovary
Uterus
Uterus
Ovary
Ovary
5.2 + 0.1
18.8 + 0.2*
14.4 0.1
69.8 4.1*
22.0 + 1.5
111.0 15.0*
ND
217.0 + 25.0*
+
+
+
All values are expressed as group means (+SEM) for the indicated
control (+R) and diabetic (dbidb) mice. All assays were run in triplicate
from pooled samples of the indicated number of animals. Diabetesrelated differences in lipase activity are denoted by a n asterisk (p <
0.05) for each tissue relative to age-matched control values (ND: not
detectable).
Fig. 4. Photomicrographs of control (A) ( X 4,630) and diabetic (B,C)
3,050) ovarian stromal cells ( S )between 8 and 16 weeks of age. The
orderly array of cells in the controls was in contrast with the rough-
(x
ened cytoplasmic membrane borders that characterized the diabetic
cells by 8 weeks of age. In 16-week-old diabetics, the cytoplasmic
contents and membranes appeared degenerative, with a n increased
intercellular (IC)space apparent between cells that was occupied by a
hyaline type of ground substance of unknown composition or origin.
366
D.R. GARRIS, R.L. WEST, AND P.H. PEKALA
utero-ovarian tissues is temporally related to the increased lipase activity in these tissues of dbfdb mice.
meextent of lipid infiltration appears to be cumulative,
the aged mice demonstrating more intracellular lipid
deposition than either younger diabetics or age-matched
controls. mesedata suppofiprevious suggestionsthat
utero-ovarian involution in these mutants may be relakd to
lipid
(Gamis et
1985c), and that the increased intracellular lipids cause
the affected cell types to exhibit metabolic activities that
are more characteristic Of adipocytes than their
(Gmis et
1985a)*It is apparent that
these structural and metabolic changes in utero-ovarian
cell types contribute to the reproductive incompetence
of this mutant murine model.
ACKNOWLEDGMENTS
The encouragement and support in terms of time,
space, and
donated by D ~Douglas
.
L. Coleman
Of these studies are greatly appreciduring the
ated. The excellent technical assistance provided by S.K.
Williams, D. Whitehead, C. Horn, and W. Bousman is
also acknowledged.
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