Journal of Environmental Protection, 2010, 1, 226-230
doi:10.4236/jep.2010.13027 Published Online September 2010 (http://www.SciRP.org/journal/jep)
Exposure to Oil during Meiosis Results in
Alterations in Meiotic Chromosomes that are
Similar to Age-Related Changes in the Nematode
Caenorhabditis elegans
Paul Goldstein
Department of Biological Sciences, University of Texas at El Paso, El Paso, USA.
Received June 16th, 2010; revised June 26th, 2010; accepted July 16th, 2010.
ABSTRACT
Exposure of young C. elegans nematodes to three different concentrations of oil resulted in changes in the meiotic
chromosomes, nucleus, nucleolus, and nuclear envelope associations. Such alterations decreased the viability and fer-
tility of this organism which was used as a biological model. The morphological changes in the “young ” group were
similar to nematodes that were senescent and post-reproductive. Comparison of meiotic chromosomes at the pachytene
stage of meiosis from young, old, and oil-exposed wild-type hermaphrodites were made following three-dimensional
electron microscopy reconstruction of serial ultrathin sections. Age-related and oil-exposure related changes included:
1) Induced condensation of chromatin with increased variance in length of chromosomes; 2) Changes in nuclear and
nucleolar volume; 3) Increased density of the nucleoplasm; and 4) Absence of Disjunction Regulator Regions, resulting
in the loss of control of the segregation of the X-chromosome into gametes during meiosis. Abnormal clustering of the
telomeric ends of the chromosomes was present on the nuclear envelope affecting the segregation of the chromosomes
during meiosis.
Keywords: Oil, Age, Chromosome, Synaptonemal Complex
1. Introduction
Cytogenetic studies in many organisms have shown that
there is a change in chromosome number or an increase
in structural chromosome alterations associated with ag-
ing [1]. Such changes would account for the cellular de-
terioration characteristic of senescent tissues since the
production of gene products would be altered. It is of
significance that such changes in normal structure and
function can be induced by exposure to oil [2].
In the present study, the free-living nematode Caenor-
habditis elegans, was exposed to varying concentrations
of oil and used as a biological model to characterize
changes associates with such exposure. This nematode is
ubiquitous and is found in virtually all types of environ-
ments, including marine. It reproduces primarily as a
self-fertilizing hermaphrodite, but a small number of
morphologically distinct males (0.3%) are also present in
the population [3]. The adult hermaphrodite has a pair of
ovaries, 810 nongonadal nuclei and five pairs of auto-
somes with two X chromosomes (2A:XX). The pachytene
nuclei are arranged peripherally around a central rachis
(Figure 1). The adult male has a single testis, 970 non-
gonadal nuclei, and five pairs of autosomes but only a
single X chromosome (2A:XO) [4]. Males arise from
gametes that have been produced after X-chromosome
nondisjunction, thus, the two sexes experience an un-
equal number of X chromosomes. They must compensate
for this state of aneuploidy and develop mechanisms for
gene expression and dosage compensation [5]. Distinct
structures on the chromosomes called “Disjunction Regu-
lator Regions” control the separation of the X chromo-
some during meiosis. Such separation is essential in or-
der to produce functional gametes that have the correct
number of chromosomes [6].
The synaptonemal complex (SC) is a tripartite, pro-
teinaceous structure that is found between paired chro-
mosomes during the pachytene stage of meiosis. It has

Exposure to Oil during Meiosis Results in Alterations in Meiotic Chromosomes that are Similar to
Age-Related Changes in the Nematode Caenorhabditis elegans
227
Figure 1. Cross-section of the gonad from C. elegans. The
meiotic nuclei (N) of the oocytes at the pachytene stage of
meiosis are arranged peripherally around a central rachis
(R). Nucleolus (Nu) Proximal arm (PA) of the gonad. Bar
equals 0.5 µm.
been highly conserved throughout evolution and occurs
in a wide variety of organisms [7]. Its presumed role is
twofold: 1) Maintenance of proximity of homologous
chromosomal segments, whereby the axial cores of the
homologs become the lateral elements of the SC [8]; and
2) Regulation of ordered meiotic disjunction in which
case the SC is maintained at the chiasma [9]. Irregular
nondisjunction of the chromosomes at anaphase I may be
a reflection of the pairing pattern at pachytene such that
multivalent associations, as in polysomies and duplica-
tions, yield complex configurations that inhibit segrega-
tion [10]. In systems in which normal bivalent formation
has been altered by the presence of chromosomal aberra-
tions, the resultant univalents do not show ordered seg-
regation patterns, and the resultant gametes are ane-
uploid.
In C. elegans, there are six SCs per germline nucleus.
The XX hermaphrodite has five SCs associated with the
autosomal bivalents and one SC for the XX bivalent. The
XO male has only five SCs since the univalent X chro-
mosome remains heterochromatic during pachytene
[11,12]. The tripartite SC consists of two lateral elements
and a striated central element with a distance between
axes of approximately 100 nm. One end of the SC is at-
tached to the inner nuclear envelope while the other end
remains free in the nucleoplasm [12]. There is no evi-
dence of the configuration known as the “bouquet”
which facilitates homologous pairing via restricted
chromosome movement [13], thus the ends of the chro-
mosomes are randomly attached around the nuclear en-
velope. In addition, there are no recombination nodules
present in C. elegans although they have been described
in other organisms [14].
Disjunction Regulator Regions (DRR) were first de-
scribed in C. elegans [11,12]. Statistical analysis of the
occurrence of DRRs and the frequency of X-chromo-
some nondisjunction support the observation that there is
a correlation between the presence of DRRs and normal
disjunction of the X chromosome [6], which is required
for the production of functional gametes.
The American Petroleum Institute’s Manifesto on
Crude Oil (CAS # 8002-05-09; (a.k.a. Sweet Crude, Arab
Medium, West Texas Inter-Cushing, Earth Oil, Petro-
leum Oil, Rock Oil, Zafiro) defines the composition of
Crude Oil as “A complex combination of hydrocarbons”.
It consists predominantly of aliphatic, alicyclic and aro-
matic hydrocarbons. It may also contain small amounts
of nitrogen, oxygen and sulfur compounds. Specifically
Crude Oil (CAS # 8002-05-9) contains Benzene, Butane,
N-Hexane, Isopentane, Pentane, and Stoddard Solvent.
Benzene is a known human carcinogen and is identified by
NTP, OSHA, and IARC as a Group 1 carcinogen. This
category encompasses light, medium, and heavy petro-
leums, as well as the oils extracted from tar sands. Along
with the numerous oil spills that are known, there are
also significant leaks and seepages into the environment
that are not reported that are equally hazardous to the
environment and negatively affect the ecosystem. As
early as 1982, cytogenetic effects, including mitotic sister
chromatid breaks and chromosome damage, were re-
ported after exposure to oil [2].
This study represents the first examination of the
changes in nuclear architecture and the meiotic chromo-
somes after exposure to oil. Serial ultrathin sections and
electron microscopy were used to provide three-dimen-
sional analysis of the relationships of all nuclear compo-
nents and identify changes correlated to the cytotoxicity
of oil.
2. Materials and Methods
The C. elegans individuals studied were hermaphrodites
received from the Caenorhabditis Genetics Center. The
worms were grown in agar plates made with 5%, 25%,
and 50% Texas Crude oil in the form of an emulsion
which they moved through at all levels of the agar. The
plate was seeded with OP50 E. coli bacteria as a food
source. In order to obtain worms at specific ages, adult
worms were removed from the plates leaving only first
stage larvae. Worms were selected at 4-5 days and 8-9
days of age, thus, forming the “young” and “old” groups
and were processed for electron microscopy as previously

Exposure to Oil during Meiosis Results in Alterations in Meiotic Chromosomes that are Similar to
Age-Related Changes in the Nematode Caenorhabditis elegans
228
described [11,15].
The pachytene karyotypes from analysis of synap-
tonemal complexes of five early-mid pachytene stage
nuclei from worms that were 4-5 days old (“young”), 8-9
days old (“old”), and 5%, 25% and 50% oil-exposed
were determined using the Metamorphosis program [16].
Nuclear and nucleolar volumes were also determined
using the Metamorphosis program. A total of 25 pachy-
tene karyotypes were analyzed.
3. Results and Discussion
Comparison of the synaptonemal complexes from the
meiotic chromosomes at the stage of pachytene, revealed
significant changes in the chromatin, the nucleus, and its
associated components (Table 1). These included: 1)
Condensation of chromatin (more condensed in older
specimens and those exposed to oil; Figure 2); 2) De-
crease in length of the chromosomes due to condensation;
3) Decrease in volume of the nucleus due to changes in
the nuclear matrix; and 4) Alterations in the association
of the telomeric ends of the chromosomes with the nu-
clear envelope, such that the normally random attach-
ment of the telomeres was restricted to a small region of
the nucleus. This resulted in a clustering of the ends of
the chromosomes, which affects their ability to segregate
properly into the gametes. In this organism, the cluster-
ing of the chromosome ends may promote entanglements
which cannot be easily resolved. In addition, the nucleus
of the old and oil-exposed individuals, accumulated elec-
tron-dense proteinaceous materials, and the nuclear en-
velope was irregular. These are manifestations of the
degradation of the nucleoplasm and protein matrix.
The synaptonemal complexes (SC), which are normally
tripartite proteinaceous structures within the meiotic
chromosomes, were altered in the ten selected worms
exposed to 25% and 50%, such that the structure of the
SCs became indistinct and the lateral elements and the
central elements were not discernible (Figure 3). This is
compared to normal SCs where the two lateral elements
were clearly visible (Figure 4). In addition, there was a
26% decrease in synaptonemal complex length compar-
ing young worms to those five selected worms exposed
to 50% oil (Figure 5, Table 1). There was a variation in
length of plus or minus of two percent from one worm to
the next. This may be the result of the abnormal conden-
sation of the chromatin which resulted in altered SC
structure. The SC is required for pairing and separation
of the chromosomes during meiosis, thus, alteration in its
structure may result in decreased viability and fecundity.
The Nuclear Volume decreased by 37% from the five
young worms and 18% from the five old worms to the
five worms selected which were exposed to 50% oil.
Figure 2. C. elegans 4-5 days old (“young”), exposed to 50%
oil, demonstrate the same condensation in chromatin (ar-
row) as in senescent worms. Bar equals 0.1 µm.
Figure 3. Synaptonemal complexes (arrow) from 4-5 day
old (“young”) C. elegans exposed to 50% oil are altered in
structure. The lateral elements and central element are not
discernible. Nuclear Envelope (NE) Chromatin (C) Bar
equals 0.2 µm.
Nuclear volume also decreased 13% between those worms
exposed to 5% versus 50% oil (Figure 6, Table 1). This
was due to abnormal condensation, i.e. heterochromati-
zation, of the chromosomes and altered protein structure.
Heterochromatization is a normal process in senescent
cells (those from C. elegans that are 8-9 days old) and
results in decreased expression of genes. The fact that the
same morphology is manifested in 4-5 day old cells
(“young”) after exposure to 50% oil, is significant, as it
indicates the deterioration and loss of function in the oo-
cytes [17].
Disjunction Regulator Regions (DRR) appeared as de-
condensed chromatin and were associated with the syn-
aptonemal complex. In C. elegans, they have been shown

Exposure to Oil during Meiosis Results in Alterations in Meiotic Chromosomes that are Similar to
Age-Related Changes in the Nematode Caenorhabditis elegans
229
Table 1. Average pachytene chromosome lengths of C. elegans wild-type from young, old, and oil-exposed individuals from
three different concentrations by reconstruction of synaptonemal complexes. All chromosome lengths are in microns.
SC # Young
(4-5 days old)
Old
8-9 days old)
Oil-Exposed
(5% Oil)
Oil-Exposed
(25% Oil)
Oil-Exposed
(50% Oil)
1x 2.5 2.4 2.4 2.2 1.9
2 4.5 4.1 4.1 4.0 3.8
3 6.0 5.5 5.3 5.0 4.7
4 6.2 6.0 6.0 5.4 4.7
5 7.9 7.1 7.0 6.8 6.5
6 10.4a 9.1a 9.0a 8.0a 6.0a
Total 37.5 34.2 33.8 31.4 27.6
Nuclear Volume µm3 12.4 9.4 9.0 8.8 7.8
Nucleolar Volume
µm3 5.6 4.5 4.5 4.4 4.2
% Nu Vol. 45.2 47.8 50.0 50.0 58.3
# DRR 6 3 0 0 0
Position of NOR
from Telomere 18% 25% 22% 19% 37%
(a) Nucleolar Organizer Region (NOR) found on this chromosome; (x) X-chromosome
Figure 4. Normal synaptonemal complex from a 4-5 day old
(“young”) C. elegans comprised of two lateral elements (ar-
row) and a striated central element that is formed between
them. Bar equals 0.1 µm.
to be responsible for controlled segregation of the X-
chromosome during meiosis [6]. The average number of
DRRs in nuclei from 4-5 day old nematodes was 6,
Figure 5. Change in meiotic chromosome length after ex-
posure to varying concentrations of oil.
whereas after they have been exposed to 50% oil, there
were none. Thus, segregation of the X-chromosome was
no longer under the proper control and unequal numbers
of chromosomes would be distributed to the gametes.
Such aneuploid gametes have decreased function.
This study was done in order to determine the effects
of exposure to oil on meiotic chromosomes and nuclear
components. The nematode C. elegans was used as a
biological model, however, the mechanisms of meiosis
are conserved throughout evolution and these aberrations

Exposure to Oil during Meiosis Results in Alterations in Meiotic Chromosomes that are Similar to
Age-Related Changes in the Nematode Caenorhabditis elegans
230
Figure 6. Change in meiotic nuclear volume after exposure
to varying concentrations of oil.
may pertain to humans as well.
4. Acknowledgements
This publication was made possible in part by Grant #
5G12RR008124 to the Border Biomedical Research
Center (BBRC)/University of Texas at El Paso from the
National Center for Research Resources (NCRR)/NIH.
The contents of this publication are solely the responsi-
bility of the authors and do not necessarily represent the
official views of NIH or NIEHS.
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