This document examines the effects of reproductive senescence on early egg development and embryonic viability in Drosophila. The main findings are: 1. Fecundity declines with age largely due to a decrease in pre-vitellogenic egg chambers and possibly retention of mature oocytes. 2. Fertility declines with age appear to result from increased abnormalities during blastoderm embryonic development, suggesting declining maternal provisioning causes developmental anomalies. 3. Embryos from old females with multiple reproductive episodes exhibited more abnormalities than those from old females with one episode, suggesting reproductive diapause may decrease but not eliminate age-related fertility decline.

1. n-­‐=11
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40
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140
160
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20
30
40
50
Reproduc)ve
senescence
nega)vely
effects
early
egg
development
and
embryonic
viability
in
Drosophila
Halie
Ostberg1,
Brian
Has3ngs1,
Claudia
Fricke2,
M.
C.
Bloch-­‐Qazi1,
1.
Department
of
Biology,
Gustavus
Adolphus
College,
St.
Peter,
MN
56082
2.
Ins3tute
for
Evolu3on
and
Biodiversity,
WesSälische
Wilhelms-­‐Universität,
Münster,
Germany
Conclusion
The
age-­‐related
decline
in
fecundity
appears
to
be
largely
due
to
a
decline
in
the
number
of
pre-­‐vitellogenic
egg
chambers
and,
possibly,
the
reten3on
of
mature
oocytes.
This
decline
may
be
due
to
a
decreasing
number
of
ovariole
stem
cells6
and
the
aging
female’s
ability
to
maintain
a
high
rate
of
egg
laying.
Female
age
does
not
appear
to
alter
the
number
of
egg
chambers,
but
has
a
borderline
nega3ve
effect
on
the
number
of
ac3ve
ovarioles.
These
measures
do
not
reflect
possible
differences
in
egg
chamber
quality
with
increasing
age.
The
age-­‐related
decline
in
fer3lity
appears
to
largely
result
from
increased
abnormal
embryonic
development
during
blastoderm
development.
This
phase
is
influenced
by
maternal
factors
sugges3ng
that
a
decline
in
maternal
provisioning
may
cause
these
developmental
anomalies.
The
embryos
of
old
females
with
mul3ple
reproduc3ve
episodes
exhibited
a
higher
percentage
of
abnormali3es
than
the
old
females
with
one
reproduc3ve
episode,
sugges3ng
that
reproduc3ve
diapause
may
decrease,
but
not
eliminate,
the
age-­‐associated
decline
in
fer3lity.
The
low
percentage
of
undeveloped
embryos
in
all
treatment
groups
suggests
that
neither
fer3liza3on
efficiency
nor
a
lack
of
ini3al
zygo3c
development
are
a
major
factors
in
the
decline
in
fer3lity.
Together,
these
results
show
that
age-­‐related
declines
in
reproduc3on
occur
at
two
dis3nct
stages
of
development:
oogenesis
and
embryogenesis.
For
each,
it
is
early
developmental
events
that
appear
to
be
the
major
determinants
of
subsequent
viability.
Acknowledgements:
We
thank
the
Gustavus
Adolphus
College
FYRE
program
for
funding
and
Dr.
Jeff
Dahlseid
for
assistance
with
confocal
microscopy
Senescence
is
the
many
mul3faceted
process
of
degrada3on
that
occurs
with
increasing
age.
In
humans
and
many
other
organisms,
aging
is
a
decrease
in
physiological
func3on
at
both
macroscopic
and
microscopic
levels.
This
func3onal
decline
can
occur
while
females
are
s3ll
reproduc3vely
ac3ve,
a
process
called
reproduc3ve
senescence.
Reproduc3ve
senescence
is
well-­‐documented
in
human
and
other
animal
popula3ons1.
Understanding
the
underlying
causes
of
it
can
lead
to
interven3ons
to
augment
human
fer3lity
and/or
control
fer3lity
in
other
animal
popula3ons.
Drosophila
melanogaster
is
a
popular
model
system
used
to
study
reproduc3ve
senescence.
Age-­‐related
declines
in
female
fecundity
(egg
laying)
and
fer3lity
(egg
hatching)
are
well
documented2.
However,
the
underlying
causes
of
reduced
fecundity
and
fer3lity
are
poorly
understood.
The
goal
of
the
present
study
is
to
refine
when
in
the
processes
of
oogenesis
and
early
embryogenesis
increasing
maternal
age
has
the
greatest
nega3ve
effects.
Experimental
Ques3ons
Why
does
fecundity
decline
with
increasing
age?
• Fewer
ovarioles
in
older
females
• Decreasing
number
of
egg
chambers
in
older
females
• Decreasing
rate
of
egg
chamber
development
in
older
females
Why
does
fer3lity
decline
with
increasing
age?
• Decreasing
fer3liza3on
efficiency
• Fewer
fer3lized
eggs
ini3ate
development
• Fewer
embryos
are
competent
to
complete
development
Results
Pre-­‐vitellogenic:
ANOVA,
fem
age
F1,44=11.080,
P<
0.0005
Vitellogenic:
F1,44=1.512,
P=.226
Post-­‐vitellogenic:
F1,44=3.156,
P=.084
Literature
Cited
1.
Finch,
C.E.
&
T.B.L.
Kirkwood.
2000.
Chance,
Development
&
Aging.
New
York:
Oxford
University
Press.
2.
Miller,
P.B.,
et
al.
2014.
Fly.
8:3,
1-­‐13.
3.
Cumings,
M.R.,
and
R.C.
King
J.
Morph.,
128:
427-­‐442.
4.
Bownes,
M.
1975
J.
Embryol.
Exp.
Morph.
Vol.33,
3,
pp.
789-­‐801.
5.
Hartenstein,
V.
1993
Atlas
of
Drosophila
Development.
New
York:
Cold
Spring
Harbor
Laboratory
Press.
6.
Pan,
L.
et
al.
2007.
Cell
Stem
Cell
1,
458-­‐469.
ANOVA,
factor
female
age,
F1,
41=3.66,
P=0.063
24
h
24
h
96
h
96
h
young
old
n-­‐=11
n-­‐=10
n-­‐=11
n-­‐=10
Ovariole
number
does
not
differ
between
young
and
old
females
Age-­‐related
embryonic
abnormali3es
peak
during
the
preblastoderm
stage
Methods
Drosophila
melanogaster
females
from
the
wild-­‐type
Dahomey
(fecundity
expts.)
and
Oregon-­‐R
(fer3lity
expts.)
strains
were
raised
and
maintained
under
standard
laboratory
condi3ons.
Females
were
collected
shortly
arer
eclosion
and
maintained
as
virgins
un3l
used
in
experiments.
Fer)lity
Females
were
aged
either
8d
(young)
or
35-­‐38d
(old)
post-­‐
eclosion
before
being
used
in
experiments.
One
group
of
old
females
(3-­‐O)
were
mated
to
young
males
weekly
during
their
adult
lives.
Young
females
(1-­‐Y)
and
the
other
group
of
old
females
(1-­‐O)
were
mated
a
single
3me
before
experiments
were
conducted.
Females
from
each
treatment
group
were
mass
mated
to
young,
wild-­‐type
males
for
24h,
then
transferred
to
oviposi3on
plates
to
collect
embryos.
Embryos
were
dechorinated
and
fixed,
then
stained
with
DAPI.
Stained
embryos
were
examined
under
epiflourescence
illumina3on
to
determine
whether
or
not
they
appeared
to
be
developing
normally
and,
if
so,
staged
by
the
number
and
arrangement
of
nuclei4,5.
Fecundity
Females
were
allowed
to
age
either
4d
(young)
or
32d
(old)
post-­‐
eclosion,
then
mated
with
wild-­‐type
males.
At
24h
arer
ma3ng,
during
peak
response
to
ma3ng
s3muli,
or
96h
arer
ma3ng,
while
the
response
to
ma3ng
is
asenua3ng,
females
were
flash-­‐frozen.
Females
were
dissected
and
we
counted
the
number
of
ovarioles,
the
total
number
of
egg
chambers,
and
staged
the
egg
chambers
using
phase-­‐
contrast
microscopy3.
Egg
chamber
development
was
grouped
into
three
stages:
pre-­‐vitellogenic,
vitellogenic,
and
post-­‐vitellogenic.
0
50
100
150
200
250
0
10
20
30
40
50
Approximately
12%
of
1-­‐Y
embryos,
32%
of
1-­‐O,
and
40%
of
3-­‐O
embryos
exhibited
abnormal
development.
65%
of
the
abnormali3es
occurred
during
the
preblastoderm
stage
of
development.
During
this
3me,
75%
of
embryos
of
old
females
with
one
reproduc3ve
episode
were
abnormal
and
84%
of
embryos
of
old
females
with
mul3ple
reproduc3ve
episodes
were
abnormal.
In
all
treatment
groups,
<3%
of
embryos
were
unfer3lized
or
failed
to
ini3ate
development.
Abnormal
Preblastoderm
Embryo
Normal
Preblastoderm
Embryo
Propor3on
of
abnormal
embryos
is
higher
in
old
females
0
50
100
150
200
250
n-­‐=154
n-­‐=225
n-­‐=216
Total
#
Embryos
1-­‐Young
1-­‐Old
3-­‐Old
%
of
embryos
at
given
phase
Abnormal
embryos
Normal
embryos
1-­‐Y
1-­‐O
3-­‐O
Preblastoderm
ovaries
n-­‐=11
n-­‐=10
n-­‐=11
n-­‐=10
young
old
24
h
96
h
24
h
96
h
24
h
24
h
24
h
24
h
24
h
24
h
96
h
96
h
96
h
96
h
96
h
96
h
young
old
old
old
young
young
pre-­‐vitellogenic
vitellogenic
post-­‐vitellogenic
Total
egg
chamber
number
does
not
differ
between
young
and
old
females
ANOVA,
factor
female
age,
F1,41=1.91,
P=0.174
Young
females
have
more
pre-­‐vitellogenic
egg
chambers
than
old
females
There
was
no
significant
difference
between
24h
and
96h
post-­‐ma3ng
treatments
for
any
of
the
variables
tested
(P>0.05).
Total
#
of
Ovarioles
(mean
+SD)
Total
#
of
egg
chambers
(man
+SD)
Total
#
of
egg
chamber
(mean
+SD)
Introduc)on
1-­‐Y
1-­‐O
3-­‐O
1-­‐Y
1-­‐O
3-­‐O
1-­‐Y
1-­‐O
3-­‐O
1-­‐Y
1-­‐O
3-­‐O
1-­‐Y
1-­‐O
3-­‐O
Blastoderm
Gastrula3on
Segmenta3on
Dorsal
Closure
Cu3cle
Specializa3on
13
48
79
12
6
3
67
6
9
2
42
31
58
90
79
23
5
16
Ovaries
Egg
chambers