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Mitochondrial alterations in embryos exposed to B-hydroxybutyrate in whole embryo culture.

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THE ANATOMICAL RECORD 213:94-lOl(1985)
Mitochondria1Alterations in Embryos Exposed
to B-Hydroxybutyrate in Whole Embryo Culture
W.E. HORTON, JR. AND T.W. SADLER
Department of Anatomy, School of Medicine, University of North Carolina, Chapel Hill,
NC 27514
ABSTRACT
The ketone body B-hydroxybutyrate (B-OHB) produces malformations and ultrastructural alterations in mitochondria of mouse embryos exposed for
24 hours to the compound in whole embryo culture. The present study was conducted
to establish the time-course of the mitochondrial changes to determine whether the
changes are reversible, and to relate these changes to the malformations produced
by the compound. Since mitochondria also play a key role in the metabolism of
ketone bodies, the capacity of the early somite embryo to metabolize B-OHB was
investigated in a n effort to link the morphological alterations in the mitochondria
to a biochemical process. Early somite embryos were cultured 4, 8, or 24 hours in
the presence of 32 mM DL-B-OHB and then cultured for a n additional 24 hours in
control serum. Finally, embryonic tissue during the teratogenic period was assessed
for its capability to oxidize B-OHB using D-(3-14C)-B-OHB.The treated embryos
showed progressive alterations in the mitochondria, beginning at 4 hours with a loss
of matrix density and culminating a t 24 hours with high-amplitude swelling, complete loss of matrix density, and disappearance of cristae. These alterations were
reversible following removal of the embryos after 24 hours of exposure to B-OHB
and culturing for a n additional 24 hours in control serum. Metabolism studies
demonstrated that the early somite embryo possesses a limited capacity to oxidatively metabolize B-OHB. The biochemical implications of these findings are discussed with respect to the possible role of ketone bodies in the mechanism of
diabetes-induced congenital malformations.
Pregnancy in the human diabetic is fraught with many
complications including a n increased incidence of congenital malformations in the offspring (Pederson, 1980).
Although many factors may be responsible for these
defects, recent evidence suggests that elevated levels of
ketone bodies (specifically B-hydroxybutyrate [B-OHB])
may play a role (McLendon and Bottomy, 1960; Drury,
1966).Two separate studies using whole embryo culture
techniques have shown that increased concentrations of
DL-B-OHB are teratogenic to both mouse (Horton and
Sadler, 1983) and rat (Lewis et al., 1983) embryos. The
embryonic response was dose and age related and was
associated with growth retardation. Histological and ultrastructural analysis of malformed embryos revealed a
single alteration in cellular morphology manifested by
high-amplitude swelling of mitochondria (Horton and
Sadler, 1983). The relationship between this alteration
and the congenital malformations is not known, although fluctuations in mitochondrial morphology have
been attributed to both normal (Hackenbrock, 1966,
1968) and pathological conditions, notably cell necrosis
(Laiho and Trump, 1975).
The mechanism by which B-OHB produces mitochondrial swelling in the intact embryo as well as the progression and time-course of the mitochondrial changes
are not known. Furthermore, the relationship between
the effects on mitochondria and cell viability has not
0 1985 ALAN R. LISS, INC
been established. Also, the ability of the mitochondria
(and whole embryos) to recover from B-OHB treatment
has not been determined. Finally, the biochemical capabilities of the early- somite embryo to oxidatively metabolize B-OHB, a process that is initiated in the
mitochondria, during the sensitive teratogenic period
has not been assessed. Therefore, the following study
was undertaken.
METHODS
Embryo Culture
Random-bred ICR mouse embryos were obtained on
the ninth gestational day (plug day = day 1)and prepared for whole embryo culture as described previously
(New, 1978; Sadler, 1979). Individual embryos (three to
five somites in age) were placed in 30-ml flasks containing 3 ml of whole, immediately centrifuged (Steele, 1972)
rat serum. Each flask was gassed for approximately 30
seconds with 5% 0 2 , 5% C02, and 90% N2 at 0 and
approximately 10 hours and placed on a rotator wheel
(30 rpm) in a 38°C incubator. For embryos maintained
longer than 24 hours, cultures were transferred to fresh
serum a t 24 hours and gassed with a 20% 0 2 , 5 % C02,
and 75% N2 mixture a t 24 and 34 hours.
Received July 16, 1984; accepted November 9, 1984.
3-OH-BUTYRATE-INDUCED CHANGES IN EMBRYOS
Upon termination of culture, embryos were examined
for gross abnormalities and analyzed for total protein
content using the Lowry method (Lowry et al., 1951) or
prepared for light and electron microscopic observation
by fixing in modified Karnovsky’s (2% glutaraldehyde,
2% paraformaldehyde), postfixing in 1% osmium tetroxide, dehydrating in alcohol and propylene oxide, and
embedding in araldite. Embedded embryos were sectioned at 1 pm with a n LKB ultramicrotome V and
stained with Toluidine blue for histological examination. In addition, thin sections were cut through cephalic
regions, stained with uranyl acetate (1%in ethanol)-lead
citrate, and examined with a JEOL 100-CX electron
microscope.
Exposure to B-OHB
A stock solution of DL-B-OHB sodium salt (Sigma
Chemical Co., St. Louis) was prepared in distilled water
and added to rat serum to achieve a final concentration
of 4 mg/ml (32 mM). This dose has previously been
shown to produce malformations and mitochondrial
swelling in a high percentage of cultured embryos (Horton and Sadler, 1983). Embryos were cultured in this
serum or in serum containing distilled water only for 4,
8, or 24 hours, then terminated and examined as described above. In addition, one group of embryos was
cultured for 24 hours in the presence of 32 mM B-OHB,
then rinsed three times in Tyrode’s solution and transferred to fresh control serum for a n additional 24 hours
of culture. A minimum of 14 embryos (seven treated and
seven controls) were cultured at each time period and
were examined with electron microscopy. Protein data
for the 48-hour time period were compared using a oneway analysis of variance.
B-OHB Metabolism
Ketone body utilization was examined on day 9 of
gestation by determining the production of I4CO2 from
D-(3-14C)B-OHB in vitro (Shambaugh et al., 1977a).
Whole conceptus (embryo and yolk sac) were pooled in
ice-cold PBS, weighed, and placed in flasks with 1.5 ml
whole, immediately centrifuged rat serum containing
4mM DL-B-OHB and 0.1 uCi/ml D-(3-14C)-B-OHB
(Amersham, specific activity = 55 mCi/mM). The tissue
was gassed with 5% 0 2 , 5% COZ 90% Nz, prior to a 4hour incubation a t 38°C. Reactions were then stopped
by placing flasks on ice and adding 0.3 ml hyamine
hydroxide to the center well and 0.3 ml 6 N Hz SO4 to
the serum and continuing to rotate the flasks for 1hour
at 38°C to evolve the CO2. The hyamine hydroxide was
then transferred from the center well to scintillation
vials containing 10 ml of Aquasol 11. The activity of the
hyamine fraction was converted to pmoles of COB
evolved from B-OHB by determining on a percent basis
the amount of labelled B-OHB utilized and assuming
and equal rate of utilization for the unlabelled compound. For comparison, maternal liver was assayed for
B-OHB utilization as described above. Each study (maternal liver and day 9 conceptus) was repeated three
times.
95
development (Figs. 1, 2). Cranial neural folds at these
time periods were in the initial stages of elevation and
consisted of a pseudostratified epithelium with three to
four nuclear layers. The luminal surface was uniform in
appearance with adjacent cells joined by terminal bars,
while a basement membrane was present on the abluminal side. Numerous mitotic figures were observed
along the luminal surface and were characterized by
spherical nuclei containing condensed chromatin. Interphase nuclei were variable in shape and contained one
to three prominent nucleoli. An occasional pyknotic cell
was present, but there was no evidence of extensive cell
necrosis or other cellular abnormalities.
Beneath the basement membrane of the neuroepithelium was a region of embryonic mesenchyme cells. Each
mesenchymal cell was composed of a nuclear region
(some in mitosis) and surrounding cytoplasmic projections which often contacted other cells. The cytoplasm of
these cells was uniform in appearance and no evidence
of cell necrosis was observed.
Ultrastructurally, neuroepithelial cells were similar
in appearance to mesenchymal and gut endoderm tissue, all resembling undifferentiated embryonic cell types
(Figs. 3,4). The cytosome of these cells was characterized
by a polysomal arrangement of the many ribosomes
present. Also observed were a few cisternae of rough
endoplasmic reticulum and a n occasional Golgi complex.
Numerous mitochondria were distributed in a random
fashion throughout the cells. These displayed distinct
inner and outer membranes, relatively few and indistinct cristae, and a homogenous dense matrix. Typical
cross sections of these organelles ranged from 0.5 to 1.0
Dm in diameter.
Four-Hour-TreatedEmbryos
After 4 hours of exposure to 32 mM DL-B-OHB, embryos displayed no gross abnormalities. Light microscopy revealed a histological picture similar to controls
in most respects (Fig. 5). The neuroepithelium was intact with a smooth luminal surface, and mesenchymal
tissue revealed no abnormal morphology and no cell
necrosis was observed. However, cytoplasmic vacuoles
were consistently observed in cells of the neuroepithelium, mesenchyme, and endoderm. These vacuoles were
spherical in shape and ranged in size from 0.5 to 1.5 pm.
They were numerous in some cells and completely absent in others.
From previous work (Horton and Sadler, 1983) it was
known that the cytoplasmic vacuoles represented mitochondria in various degrees of swelling. Therefore, electron microscopic examination of 4-hour-treated embryos
was performed to characterize the altered mitochondrial
morphology. In general, most of these organelles appeared similar to controls with a dense matrix and relatively indistinct cristae. At the other extreme,
mitochondria were observed that showed definite swelling and these organelles displayed a pale homogenous
matrix. In some cases cristae were present (more distinct
against the pale matrix than in controls) while other
examples displayed few or no cristae. A third morpholRESULTS
ogy was observed at this time period, in which the miFour- and Eight-Hour Control Embryos
tochondria appeared in the initial stages of swelling
Control embrvos examined with light microscopy a t 4 (Fig. 6). These organelles displayed a dense matrix interand 8 hours oiculture displayed a-similar patiern of sp&sed with isdated areas of pale matrix, although
96
W.E. HORTON, JK.AND T.W. SADLER
Fig. 1. Cross section through an early somite embryo cultured 4
hours in control serum. NF, cranial neural fold; H, heart; YS, visceral
yolk sac. x23.
Fig. 3. Portion of a neuroepithelial cell from an embryo cultured 4
hours in control serum. Numerous mitochondria (M) cut in cross section and longitudinally are dispersed throughout the cell. x 19,800.
Fig. 2. Neuroepithelium from embryo cultured 4 hours in control
serum (a similar morphology is present after 8 hours in culture). The
neuroepithelium WE) is psuedostratified with three to four nuclear
layers. Mitotic figures are observed (arrow) and no abnormal cytoplasmic structures are evident. A, amnion; M, mesenchyme. ~ 9 0 0 .
Fig. 4. High-magnification view of typical mitochondria from a control embryo. Note the dense matrix and the relative indistinct appearance of the cristae (C). Also, observe the polysomal arrangement of the
ribosomes (P). ~40,700.
Fig. 5. Neuroepithelium (NE) from an embryo cultured 4 hours in
the presence of 32 mM DL-B-OHB. Note the presence of numerous,
small, cytoplasmic vacuoles (arrows). A, amnion; M, mesenchyme.
x660.
Fig. 8 . Electron micrograph of a neuroepithelial cell from the embryo
described in Figure 7. Note the swollen mitochondria (M).C,cristae; P,
polysomes. x 30,500.
Fig. 6. Electron micrograph of a neuroepithelial cell from the embryo
described in Figure 5. Note the electron-lucent portions of the mitochondrial matrix (arrows). P, polysomes. x 30,500.
Fig. 9. Neuroepithelium (NE) from a n embryo cultured 24 hours in
the presence of 32 mM DL-B-OHB. Cytoplasmic vacuoles are widespread and many are larger than those observed at earlier time periods
(arrows). x660.
Fig. 7. Neuroepithelium (NE) from an embryo cultured 8 hours in
the presence of 32 mM DL-B-OHB.Cytoplasmic vacuoles (arrows) are
prevalent. “Blebbing” of the luminal surface of the neuroepithelium
has occurred (arrowheads, compare to Fig. 5). X660.
Fig. 10. Electron micrograph of a neuroepithelial cell from the embryo described in Figure 9. A single, highly swollen mitochondrion is
present (M) with a pale matrix and reduced cristae (C). P, polysomes.
~30,500.
98
W.E. HORTON, JR.AND T.W. SADLER
there was no apparent increase in the overall size of
these structures. No other ultrastructural abnormalities
were observed at this time period.
Eight-Hour-TreatedEmbryos
Electron microscopy was performed on 48-hour control
and treated tissue to assess the degree of recovery of
mitochondria after exposure to DL-B-OHB. Interestingly, both control and treated tissue displayed similar
profiles of mitochondrial morphology (Figs. 13, 141, although the appearance was distinct from that observed
in control embryos a t earlier time periods. The control
and “recovered” mitochondria each displayed a prominent inner and outer membrane. The inner membrane
continued into the matrix region as cristae, which appeared more distinct than a t earlier time periods. Matrix morphology was also different from control tissue
cultured for 4 and 8 hours. For example, earlier the
matrix was electron-dense, whereas mitochondria from
embryos (treated and controls) cultured for 48 hours
exhibited a pale matrix consisting of a fine, homogeneous material. This pattern was especially evident in
mitochondria that had been cut in a longitudinal profile.
As with the earlier time period, no gross abnormalities
were observed in embryos exposed 8 hours to DL-BOHB. At the light microscopic level, the most striking
finding was a n increase in the size and number of cytoplasmic vacuoles. These structures were now present in
virtually every cell and in some cases reached 2 pm in
diameter (Fig. 7). Also, a change in the appearance of
the luminal surface of the neuroepithelium was observed. Instead of the smooth configuration observed in
4-hour-treated embryos and all controls, the luminal
surface was now irregular and characterized by “blebbing” of the surface cytoplasm.
Electron microscopy revealed a progression in the mitochondrial response to B-OHB compared to the 4-hour
Metabolism of B-OH6
time period. The majority of the mitochondria now disThe
ability
of
developing
tissue to oxidatively metabplayed high-amplitude swelling with loss of matrix density and few identifiable cristae (Fig. 8). In spite of this olize B-OHB to GO2 on day 9 of gestation was detersevere alteration in mitochondrial morphology, no other mined. At this stage (three to five somites), the mouse
conceptus produced 0.84 & 0.26 pM COz/g wet weight/
ultrastructural changes were observed.
minute from B-OHB. This value was slightly higher
Twenty Four-Hour-Treated Embryos
than the level determined for maternal liver (0.64 k
Embryos cultured 24 hours in 32 mM DL-B-OHB dis- 0.34 pM C02/g wet weightlminute) which is known to
played gross defects, as described previously (Horton possess only a slight capacity to metabolize the comand Sadler, 1983). These included cephalic and caudal pound (Mahler, 1953) and, therefore, served as a referneural tube closure defects, abnormal rotation, and ence for comparison with embryonic metabolic rates.
growth reduction. Light histology revealed only minimal cell necrosis, and the presence of widespread cytoDISCUSSION
plasmic vacuoles throughout the tissue (Fig. 9).
The
present
study
represents
a n extension of previous
Ultrastructurally these swollen mitochondria appeared
more severely affected than at the 8-hour time period work dealing with interactions between the ketone body,
(Fig. 10). The majority of the organelles were widely B-OHB, and the developing embryo in vitro. The results
dilated (up to 3 pm in diameter) with a n extremely pale show that high concentrations of DL-B-OHB (32 mM)
matrix. For the most part, normal cristae were not ap- induce progressive mitochondrial changes in mouse emparent. As with all other time periods, the polysomal bryos growing in whole embryo culture. Specifically,
arrangement of ribosomes was intact and no other ultra- mitochondria undergo high-amplitude swelling, which
begins by 4 hours of exposure to B-OHB and becomes
structural abnormalities were noted.
increasingly pronounced during the 24-hour culture peForty Eight-Hour Recovery Studies
riod. This swelling occurs in the absence of ultrastrucControl embryos cultured for 48 hours were similar in tural alterations in other cellular organelles and is
appearance to comparably staged in vivo embryos (Fig. reversible if embryos are cultured for a n additional 24
11).These embryos possessed 29-31 somites, had ad- hours in control serum.
The specific mechanism by which B-OHB produces
vanced forelimb development, and displayed a completely closed neural tube. Furthermore, the cranial mitochondrial swelling in the intact embryo is not
region showed brain vesicle development resulting in known. Mitochondria have been shown to undergo morsubdivision of the neural tube into forebrain, midbrain, phological alterations in response to a variety of physiological, pathological, and physical conditions. For
and hindbrain.
Embryos cultured for 24 hours in the presence of 32 example, isolated mitochondria behave as osmometers,
mM DL-B-OHB, followed by 24 hours in control serum shrinking and swelling in response to either a hyperdisplayed abnormal development compared to controls tonic or hypotonic medium, respectively (Lehninger,
(Fig. 12). The majority of the treated embryos (92%)had 1962). In this regard, the addition of 32 mM levels of Bcompleted cranial neural tube closure and averaged 28- OHB raises the serum osmolarity approximately 40
30 somites. However, 50%(13/26)of the embryos showed mOsmAiter, i.e. from 300 to 340. However, this increase
lack of closure of the posterior neuropore. Also, treated is not significant considering that embryos cultured 24
embryos consistently failed to undergo normal brain hours in the presence of 64-mM levels of L-glucose (which
vesicle formation. These embryos displayed a rounded produces serum osmolarity levels in excess of 340 m O s d
head with lack of expansion of the prosencephalon. Fi- liter) grow normally and show no cytoplasmic vacuoles
nally, treated embryos were growth reduced, averaging (unpublished results). These results confirm earlier work
193 f 32 pg (N = 13)protein compared to 312 k 69 k g which demonstrated that high concentrations of L-glucose which produced osmolarities in excess of 390 mOsm/
(N = 12)protein for controls (P < .01).
3-OH-FUTYRATE-INDUCEDCHANGES IN EMBRYOS
99
Fig. 11. An embryo cultured 48 hours in control serum. H, heart; L,
forelimb; 0, otic vesicle; P, prosencephalon; M, mesencephalon; R,
rhombencephalon. ~ 2 6 .
Fig. 13. Typical mitochondria from an embryo cultured 48 hours in
control serum. Compare to Figure 4 and note paler matrix and more
distinct cristae (C). P, polysomes. x 39,000.
Fig. 12. An embryo cultured 24 hours in the presence of 32 mM DLB-OHB and then 24 hours in control serum. The embryo shows an
overall growth reduction and retarded brain development, especially
noticeable by the lack of prosencephalic expansion. X26.
Fig. 14. Typical mitochondria from an embryo exposed to 32 rnM BOHB and then transferred to control serum for an additional 24 hours.
The mitochondria1 morphology appears similar to that observed in
controls with a pale matrix and distinct cristae (C). P, polysomes.
x 39.000.
100
W.E. HORTON, JR.AND T.W. SADLER
liter were not teratogenic to cultured rat embryos (Cockroft and Coppola, 1977).
It is also possible that the passage of DL-B-OHB into
the embryo and, ultimately, the mitochondria results in
fluid movement and mitochondrial swelling. Yet, the
time course of the swelling as revealed by electron microscopy (occurring somewhere between 4 and 8 hours)
would seem too slow for what should be a rapid osmotic
event. Also, fluid movement into the cell would be expected to cause more general morphological alterations,
such as dilution of the cytosol, dilation of endoplasmic
reticulum, or, possibly, nuclear changes. However, the
ultrastructural studies demonstrated no differences between control and treated tissue except for the mitochondria] alterations.
In addition to osmotic responses, mitochondrial morphology is also influenced by fixation. For example, low
concentrations of paraformaldehyde (< 1%)have been
shown to induce mitochondrial swelling in liver tissue
(Romert and Matthiessen, 1981). However, it is unlikely
that the results observed in this study are due to nonspecific effects of fixation. The concentration of paraformaldehyde used in this study was well above the level that
produces swelling and, in fact, no abnormal mitochondria were noted in control tissues which were processed
simultaneously with treated specimens. Also, embryos
exposed to B-OHB and then fixed in 4% osmium tetroxide only displayed the cytoplasmic vacuoles indicative of
mitochondrial swelling (unpublished results).
Several investigators have observed high-amplitude
mitochondrial swelling in association with cell injury
and cell death (Laiho and Trump, 1975).Although some
cell necrosis is visible after 24-hour exposure to B-OHB,
the mitochondrial change does not seem to be linked to
cell death. Mitochondrial swelling occurs in the majority
of cells of the embryo (dying and non-dying), whereas
only minimal cell necrosis is observed. Also the mitochondrial change occurs in the absence of other ultrastructural markers for cell death such as dispersal of
polysomes (Sadler and Cardell, 1977). Finally, the mitochondrial swelling is fully reversible if B-OHB is removed from the culture medium.
Mitochondrial changes induced by B-OHB might also
have a biochemical basis. The enzyme pathway for the
oxidative metabolism of ketone bodies is intramitochondrial, involving the conversion of B-OHB to acetoacetate
by the stereospecific enzyme B-OHB dehydrogenase (Robinson and Williamson, 1980). It has been postulated
that the shunting of B-OHB through this metabolic
pathway decreases intramitochondrial NAD+/NADH
ratios and may lead to alterations in mitochondrial function (Goldstein et al., 1982). The present study has demonstrated that the early somite embryo can metabolize
B-OHB to C 0 2 a t a rate slightly greater than that obtained for maternal liver which minimally utilizes ketone bodies (Mahler, 1953). Thus, it is possible that BOHB is undergoing initial stages of metabolism leading
to alterations in redox potential of the mitochondria.
In addition to their association with mitochondria, BOHB andor its metabolites are known to interact with
several other biochemical pathways, including inhibition of (1)uptake and utilization of glucose by adult and
fetal tissues (Robinson and Williamson, 1980; Shambaugh et al., 197713); (2) the oxidative metabolism of
alpha-ketoisocaproic acid (Shambaugh and Koehler,
1983);and (3)pyrimidine synthesis in the fetal rat brain
(Bhasin and Shambaugh, 1982). This last interaction
has been implicated as a potential mechanism by which
ketone bodies might inhibit cell proliferation and lead
to the lowered brain weight in offspring of women experiencing ketonemic states late in pregnancy (Bhasin and
Shambaugh, 1982).
In the present study, embryos cultured for 24 hours in
B-OHB and 24 hours in control serum displayed abnormal brain development with lack of brain vesicle formation, and were growth reduced. Also, mitotic indices
performed on embryos treated 24 hours with 32 mM DLBOHB revealed a 30% decrease in mitotic rate (unpublished results). Thus, it is possible that the effects of BOHB on the developing embryo may be mediated via a n
effect on cell proliferation.
Studies are currently underway in our laboratory to
further assess the effects of B-OHB on pyrimidine metabolism and to determine the effects of acetoacetate on
embryogenesis and mitochondrial morphology. It is
hoped that these types of studies will lead to a better
understanding of possible biochemical alterations produced by ketone bodies in the developing embryo and,
thus, clarify potential mechanisms of diabetes-induced
congenital malformations.
ACKNOWLEDGMENTS
This work was supported in part by the Kroc Foundation, the Ryan Foundation, and NIH grant #HD 17381.
The authors greatly appreciate the technical assistance
of Julie Yonker and Sheryl Tulis, and the typing assistance of Robin Wynn.
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