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Improving C
umulus-O
ocyte-Com
plex Yield in a Superovulation Protocol for Perom
yscusm
iceR
achael Labitt 1,2
and YukselA
gca1
1De
pa
rtme
nt o
f Ve
terin
ary P
ath
ob
iolo
gy, C
olle
ge
of V
ete
rina
ry Me
dicin
e, U
nive
rsity of M
issou
ri, Co
lum
bia
, MO
2Co
lleg
e o
f Ve
terin
ary M
ed
icine
, Co
rne
ll Un
iversity, Ith
aca
, NY
•T
he Perom
yscusgenus of m
ice, or deer m
ice, are used in research on evolution, gene im
printing, monogam
y, and environm
ental toxicology.•
Preserving and resurrecting strain variants or
generating new transgenic lines is im
possible w
ith current protocols, and superovulation protocols for M
usm
ice are ineffective.•
Previous superovulation studies in
Perom
yscushave yielded sim
ilar numbers of
cumulus-oocyte-com
plexes (CO
Cs) to that
which a fem
ale would naturally ovulate, or
require cumbersom
e in vitro maturation
techniques. 1
•T
he purpose of the present study was to
optimize an efficient, scalable superovulation
protocol for deer mice.
•S
ince the timing of P
eromyscus
follicular w
aves is unknown, w
e hypothesized that a sustained release of F
SH
via osmotic pum
p w
ould increase CO
C yield com
pared to previously published injection m
ethods.
t=56 hrsIn
du
ceo
vula
tion
Co
un
t o
varia
n
follicle
s
Su
bcu
tan
eo
us im
pla
nt:
5 mg FSH
viao
smo
tic pu
mp
5 IU PM
SG
intra
pe
riton
ea
lly
Perom
yscusm
aniculatusF
em
ale
s ag
es 11
-25
we
eks
Implant group
Control group
t=0 hrsIn
du
cefo
llicula
rd
eve
lop
me
nt
Animals
t=71 hrsO
vidu
cth
arve
st
Co
un
t CO
Cs
5 IU H
CG
5 IU LH
10 IU LH
AB
CD
Figure 1. Pum
p effect on ovarian folliculogenesis.O
varies were harvested at 56 hours from
Perom
yscusaged 11-15 w
eeks that had rece
ived
5 IU
PM
SG
I.P. (n=1
0) o
r an o
smotic pum
p conta
ining 5
mg F
SH
(n=9). (A)T
he number of follicles per ovary are sim
ilar between
groups (p=0.92). (B
) Two ovaries from
Perom
yscusthat received F
SH
via osmotic pum
p showing m
ature follicles, which w
ere larger and m
ore uniform than the control P
MS
G group (data not show
n). (C) and
(D)
Oocyte and zona
pellucida(arrow
) are visible within follicles of
control PM
SG
group (C) o
r FS
H pum
p trea
ted
(D) m
ice.
AB
Figure 2. Morphology of C
OC
s. CO
Cs w
ithin the oviduct (A)or after
dissection into media (B
). CO
Cs form
an elongated structure in the oviduct that is rem
oved by an action similar to “squeezing a tube of
toothpaste”, unlike the clustered clutch produced by Mus
mice. T
his suggests that superovulation is not a sim
ultaneous event in P
eromyscus.
Figure 3.O
ptimizatio
n of CO
C yield. F
SH
implant follow
ed by 10 IU LH
yields more C
OC
s. (A)C
OC
yield by treatment group (n=
15). (B)
CO
C yield stratified by age group (n=
5). Perom
yscus17-20 w
eeks old treated with F
SH
osmotic pum
p and 10 IU LH
yield the most oocytes,
significantly higher than the conditions previously published, 12-14 week old m
ice treated with 5 IU
PM
SG
and 5 IU H
CG
.(C) S
uperovulation success rate, stratified by age group (n=
5). Success w
as defined as the percent of mice that produced ≥6 C
OC
s and therefore more than
one standard deviation above our colony’s average litter size of 4.2+/-1.5 pups.
AB
BC
•M
any unovulatedfollicles
remained and ovulation did not
appear synchronous; higher yields m
ay be achieved by w
aiting longer before CO
C
collection.•
We did not test the 13-15 w
eek tim
e point, which m
ay be the m
ost effective age. •
This efficient, practical m
ethod of superovulation is the first step tow
ard eventual embryo
transfer protocols or transgenic m
anipulation of Perom
yscus.
Methods
Results
Introduction
Conclusions
1.
Ch
oi JK
, He
X (2
01
3) In Vitro
Ma
tura
tion
of C
um
ulu
s-Oo
cyte
Co
mp
lexe
s for E
fficien
t Isola
tion
of O
ocyte
s from
Ou
tbre
d D
ee
r M
ice. P
Lo
SO
NE
8(2
): e5
61
58
. do
i:10
.13
71
/jou
rna
l.po
ne
.00
56
15
8
References
Ka
itlin E
pp
erso
n fo
r hu
sba
nd
ry sup
po
rt. Re
sea
rch G
ran
t: NIH
-Mu
tan
t Mo
use
R
eso
urce
Ce
nte
r. Stu
de
nt S
up
po
rt: Gla
xoS
mith
Klin
e, A
me
rican
So
ciety fo
r L
ab
ora
tory A
nim
al P
ractitio
ne
rs Fo
un
da
tion
, IDE
XX
-Bio
Re
sea
rch
Acknowledgem
ents
New
protocolPreviously publishedconditions
Animal age
17-20 weeks
12-14 weeks
First hormone
5 mg F
SH
via subcutaneous osm
otic pump im
plant5
IU P
MS
G
Second hormone
10 IU LH
i.p., t=56 hours
5 IU H
CG
, t=56 hours
CO
C collection
t=71
hours (needs optim
ization)t=
71 hours
Average CO
C yield
9 (range 5-15)4.2 (range 0-5)
Superovulation success rate
80%0%
Statistical analysis: Data are plotted as m
ean +
/-S
D. H
istograms w
ere plotted (not shown) to
determine that data w
as non-parametric.
Dichotom
ous data was analyzed by M
ann-W
hitney test (Fig. 1A
, Fi. 3B
) with B
onferronicorrection for m
ultiple tests (Fig. 3B
). P
olychotomous
data was analyzed by K
ruskal-W
allis test (Fig. 3A
). * p<0.05.
New
efficient conditions for superovulation of Peromyscus
mice